Modified relaxin polypeptides and their uses

ABSTRACT

Modified relaxin polypeptides and their uses thereof are provided. Exemplary embodiments provide relaxin polypeptides which include one or more amino acid substitutions with natural or non-naturally encoded amino acids, and/or linkage to a water-soluble polymer, such as polyethylene glycol. Additionally, use of said relaxin polypeptides for treatment of disease, such as heart failure, is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/884,639filed May 27, 2020, which is a divisional of U.S. Ser. No. 16/285,986,filed Feb. 26, 2019, now U.S. Pat. No. 11,311,605, which is a divisionalof U.S. patent application Ser. No. 15/386,347, filed Dec. 21, 2016, nowU.S. Pat. No. 10,253,083, which is a divisional of U.S. patentapplication Ser. No. 13/774,349, filed Feb. 22, 2013, now U.S. Pat. No.9,567,386, which is a continuation-in-part of U.S. patent applicationSer. No. 13/212,101, filed Aug. 17, 2011, now U.S. Pat. No. 8,735,539,which claims benefit to U.S. Provisional Pat. Appl. No. 61/374,582,filed Aug. 17, 2010, each of which is hereby incorporated by referencein its entirety.

SEQUENCE LISTING

This application includes a sequence listing which has been submittedvia EFS-Web in a file named “1143270o001211.txt” created Mar. 3, 2022,and having a size of 54,192 bytes, which is hereby incorporated byreference in its entirety.

FIELD

The present disclosure relates to relaxin polypeptides and uses thereof.In exemplary embodiments, the relaxin polypeptides, such as humanrelaxin-2 polypeptides, may include one or more amino acid modificationsand/or post-translational modifications, such as linkage to apolyethylene glycol or other half-life extending molecule. Thedisclosure further provides pharmaceutical compositions and medical useof such relaxin polypeptides.

BACKGROUND

Human relaxin 2 (RLX-2, H2-RLX, H2, RLXH2, bA12D24.1.1, bA12D24.1.2,hR2, RLN2) is a member of the insulin peptide family which includes therelaxins (RLX-1, RLX-2, RLX-3), insulin, and a number of insulin likepeptides (INSL3-6), and the insulin-like growth factors (IGFI and IGFII)(Van Der Westhuizen et al., Curr Drug Targets, 2007. 8(1): p. 91-104).The active form of the protein consists of an A chain and a B chainlinked by disulfide bonds. Relaxin is produced by the ovary, and targetsthe female reproductive system to ripen the cervix, elongate the pubicsymphysis and inhibit uterine contraction. (Sherwood et al., Endocr Rev,2004. 25(2): p. 205-34.)

Mature human relaxin is a hormonal peptide of approximately 6000 daltonsknown to be responsible for remodeling the reproductive tract beforeparturition, thus facilitating the birth process. This protein appearsto modulate the restructuring of connective tissues in target organs toobtain the required changes in organ structure during pregnancy andparturition. See, Hisaw, F. L., Proc. Soc. Exp. Biol. Med., 23: 661-663(1926); Schwabe, C., et al., Biochem. Biophys. Res. Comm., 75: 503-570(1977); James, R. et al., Nature, 267: 544-546 (1977). A concise reviewof relaxin was provided by Sherwood, D. in The Physiology ofReproduction, Chapter 16, “Relaxin”, Knobil, E. and Neill, J., et al.(eds.), (Raven Press Ltd., New York), pp. 585-673 (1988). Circulatinglevels of relaxin are elevated for the entire nine months of pregnancyand drop quickly following delivery.

While predominantly a hormone of pregnancy, relaxin has also beendetected in the non-pregnant female as well as in the male(Bryant-Greenwood, G. D., Endocrine Reviews, 3: 62-90 (1982) and Weiss,G., Ann. Rev. Physiol., 46:43-52 (1984)) and has most recently beenfound to be useful in the treatment of heart failure.

Relaxin has been purified from a variety of species including porcine,murine, equine, shark, tiger, rat, dogfish and human, and shows at leastprimary and secondary structural homology to insulin and theinsulin-like growth factor, however homology between species can bequite low. In the human, relaxin is found in most abundance in thecorpora lutea (CL) of pregnancy. However, specific nuclei in the brainhave relaxin receptors and other nuclei contain messenger RNA forrelaxin. Several nuclei with cells bearing relaxin receptors are foundin the area of the hypothalamus.

Two human gene forms have been identified, (H1) and (H2). Hudson, P., etal., Nature, 301: 628-631 (1983); Hudson, P., et al., The EMBO Journal,3: 2333-2339 (1984); and U.S. Pat. Nos. 4,758,516 and 4,871,670. Onlyone of the gene forms (H2) has been found to be transcribed in CL. Itremains unclear whether the (H1) form is expressed at another tissuesite, or whether it represents a pseudo-gene. When synthetic humanrelaxin (H2) and certain human relaxin analogs were tested forbiological activity, the tests revealed a relaxin core necessary forbiological activity as well as certain amino acid substitutions formethionine that did not affect biological activity. Johnston, et al., inPeptides: Structure and Function, Proc. Ninth American PeptideSymposium, Deber, C. M., et al. (eds.) (Pierce Chem. Co. 1985).

A third relaxin gene form, Human relaxin-3 (also called H3 relaxin,RLX-3) is the equivalent to relaxin-2 in other species and is moderatelyconserved across species (amino acid homology of 77% between mouse andhuman) (Bathgate et al., J Biol Chem, 2002. 277(2): p. 1148-57). Becauseof its high expression levels in brain, human RLX-3 is considered aneuropeptide.

Methods of making relaxin are also described in U.S. Pat. No. 4,835,251and in U.S. Pat. No. 5,464,756, international publicationWO/1990/013659, U.S. Ser. No. 08/080,354, U.S. Pat. No. 5,759,807, andinternational publication no. WO/1995/000645. Methods of using relaxinin cardiovascular therapy and in the treatment of neurodegenerativediseases are described in U.S. Pat. No. 5,166,191 and in U.S. Ser. No.07/902,637 (related to publication WO/1993/003755). Certain formulationsof human relaxin are described in U.S. Pat. No. 5,451,572.

Recombinant human relaxin (H2) has been in Phase I human clinical trialsin scleroderma patients. Scleroderma is a disease involving an imbalancein tissue reformation giving rise to the overproduction of collagen, andultimately resulting in swelling and hardening of the skin (and affectedorgans). Currently treatments delivering relaxin require repeated andprolonged infusions.

Heart failure (HF) is defined as the inability of the cardiac pump tomove blood as needed to provide for the metabolic needs of body tissue.Decreases in pumping ability arise most often from loss or damage ofmyocardial tissue. As a result, ventricular emptying is suppressed whichleads to an increase in ventricular filling pressure and ventricularwall stress, and to a decrease in cardiac output. As a physiologicalresponse to the decrease in cardiac output, numerous neuroendocrinereflexes are activated which cause systemic vasoconstriction,sympathetic stimulation of the heart and fluid retention. Although thesereflex responses tend to enhance cardiac output initially, they aredetrimental in the long term. The resulting increases in peripheralresistance increase the afterload on the heart and the increases inblood volume further increase ventricular filling pressure. Thesechanges, together with the increased sympathetic stimulation of theheart, lead to further and often decompensating demands on the remainingfunctional myocardium.

Congestive heart failure, which is a common end point for manycardiovascular disorders, results when the heart is unable to adequatelyperfuse the peripheral tissues. According to an estimate published in1987, there were about 4 million people in the United States diagnosedwith this disease, and more than 50% of these cases are fatal within 5years of diagnosis (Taylor et al., Annual Reports in Med. Chem. 22,85-94 (1987)).

A more recent report indicates that heart failure continues to representa tremendous burden on today's health care system with an estimatedUnited States prevalence of 5.7 million (Roger et al., Circulation,2012. 125(1): p. e2-e220). It is further estimated that by 2030, anadditional 3 million people will have HF, a 25.0% increase from 2010(Heidenreich, et al., Circulation, 2011. 123(8): p. 933-44). Theestimated direct costs (2008 dollars) associated with HF for 2010 was$25 B, projected to grow to $78 B by 2030 (id.). While survival after HFdiagnosis has improved over time (Matsushita et al., Diabetes. 59(8): p.2020-6; Roger et al., Jama, 2004. 292(3): p. 344-50), the death rateremains high with ˜50% of people with HF dying within 5 years ofdiagnosis (Roger et al. (2012), supra; Roger et al. (2004), supra).

Current therapy for heart failure, including congestive heart failure,focuses on increasing cardiac output without causing undue demands onthe myocardium. To achieve these ends, various combinations ofdiuretics, vasodilators and inotropic agents are used to decrease bloodvolume, to decrease peripheral resistance, and to increase force ofcardiac contraction. Current therapy therefore depends on balancing theeffects of multiple drugs to achieve the clinical needs of individualpatients, and is plagued by adverse reactions to the drugs used.

For example, diuretics decrease plasma concentrations of potassium andmagnesium and increase the incidence of arrhythmias in patientsreceiving digitalis. Diuretics can potentiate the circulatory effects ofnitrates through volume depletion and lead to decreases in fillingpressure of the heart, cardiac output and systemic arterial pressure.

Alpha adrenergic antagonists can lead to marked falls in systemicarterial pressure that compromise coronary perfusion.

Angiotensin converting enzyme inhibitors can have similar effects onarterial pressure and additionally lead to excessive increases in plasmaconcentrations of potassium.

Drugs that lead to positive inotropy, such as digitalis (digoxin) andbeta adrenergic antagonists, have the potential to provoke arrhythmias.In addition, digitalis has a narrow therapeutic index and thecatecholamine analogs all tend to lose their effectiveness rapidly, dueto receptor downregulation.

Thus there is a need for therapeutic agents that lead to physiologicallyintegrated responses of arterial and venous vasodilation and cardiacinotropy, and are devoid of the disadvantages of the currently usedtherapeutic agents.

SUMMARY

The present invention provides modified relaxins, as well ascompositions and therapeutic uses thereof.

One such modified relaxin is RLX-A-AQ1-20KPEG, which relative to humanwild-type relaxin 2 is substituted with a natural and a non-naturallyencoded amino acid, and additionally is linked to a 20 kDA polyethyleneglycol (PEG). As further described in the examples below.RLX-A-AQ1-20KPEG is demonstrated to retain properties of wild-typerelaxin that are indicative of clinical efficacy, including selectivereceptor activation, anti-fibrotic activity, and efficacy in a rodentmodel of heart failure. Significantly, the in vivo half-life ofRLX-A-AQ1-20KPEG is demonstrated herein to be greatly increased comparedto wild-type.

In an exemplary embodiment, the modified relaxin may include at leastone non-naturally encoded amino acid. In exemplary embodiments, themodified relaxin may be linked to a water soluble polymer, such as apolyethylene glycol (PEG). Said PEG may have a molecular weight of about1 kDA, 2 kDA, 3 kDA, 5 kDa, 10 kDA, 15 kDa, 20 kDA, 25 kDA, 30 kDA, 35kDA, or 40 kDA, or more, or any range therein, e.g., between about 1-40kDA, between about 2-35 kDA, between about 5-30 kDA, between about 15-25kDA, or between about 17-23 kDA.

The water soluble polymer may be covalently linked to said modifiedrelaxin, e.g., covalently linked to a non-naturally encoded amino acid.For example, the modified relaxin may comprise a PEG linked to ap-acetylphenylalanine via an oxime linkage.

In exemplary embodiments, the in vivo half-life of the modified relaxinis increased compared to wild-type, such as an in vivo half-life of atleast 1, 2, 5, 10, 12, or 15 hours. For comparison, the half-life ofintravenously administrated Relaxin 2 in humans is less than 10 minuteswhen administered IV (Dschietzig T. et al., Pharmacol. Ther. 112:38-56,2009).

In an exemplary embodiment, the disclosure provides RLX-A-AQ1-20KPEGpolypeptide, having the A chain of SEQ ID NO:35, the B chain of SEQ IDNO:6, which A and B chains are linked by the two interchain disulfidebonds as in wild-type relaxin (i.e. between position 11 of the A-chainand position 11 of the B-chain, a second between position 24 of theA-chain and position 23 of the B-chain) and which A chain includes theintrachain disulfide bond of wild-type relaxin (i.e., between positions10 and 15 of the A-chain), wherein the pACF at position 1 in the A chainis linked to a 20 kDA PEG via an oxime linkage.

In another exemplary embodiment, the disclosure provides one or morepolynucleotides encoding the A and B chains of RLX-A-AQ1-20KPEGpolypeptide or another modified relaxin of the disclosure. Thenon-naturally encoded amino acid (such as pACF) may be encoded thereinby a selector codon (such as an amber codon, ochre codon, opal codon, aunique codon, a rare codon, a five-base codon, or a four-base codon)which may be recognized by an orthogonal tRNA comprising saidnon-naturally encoded amino acid, thereby permitting incorporation ofsaid non-naturally encoded amino acid during translation. Said A and Bchain-coding polynucleotides may be contained in different molecules orin the same molecules, e.g., joined in any order and optionally joinedvia a connecting peptide. Optionally said connecting peptide comprises,consists of, or consists essentially of residues 30-64 of SEQ ID NO: 3or a natural prorelaxin connecting peptide sequence, e.g., those foundin SwissProt accession no. P04090 namely the sequences SLSQEDAPQTPRPVAEIVPSFINKD TETINMMSEF VANLPQELKL TLSEMQPALP QLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKY LGLDTHS (SEQ ID NO: 43) or the sequenceSLSQEDAPQTPRPVA EIVPSFINKD TETINMMSEF VANLPQELKL TLSEMQPALPQLQQHVPVLKDSSLLFEEFK KLIRNRQSEAADSSPSELKY LGLDTHSRKK R (SEQ ID NO: 44)or optionally including any of the natural variant sequences describedthereof. Said polynucleotide may be isolated. Said polynucleotide may becontained in one or more vectors, plasmids, etc., such as an expressionvector. Additional exemplary embodiments provide a composition fortranslation of said polynucleotides, such as a cell or in vitrotranslation system comprising said polynucleotides. Further exemplaryembodiments provide a host cells, such as a prokaryotic cell (e.g., E.coli) or eukaryotic cell (e.g., a yeast or mammalian cell) comprisingsaid polynucleotide and optionally further comprising said orthogonaltRNA. Additional exemplary embodiments provide a method of producing amodified relaxin, comprising causing a cell or in vitro translationsystem to translate said polynucleotide or an mRNA transcribedtherefrom.

Optionally, the modified relaxin may include one or more natural variantsequences of human relaxin 2, such as M28K (variants rs2020050), asdefined in human prorelaxin 2 sequence of SwissProt accession no.P04090, i.e., substitution of lysine for methionine in the fourthposition in SEQ ID NO:3, 5, or 6 (within the B chain). Additionalnatural variant sequences which may be present include sequences in thesignal sequence and linker of SwissProt P04090, including thesubstitutions P68Q or V12F (rs2020050 and rs2273783, respectively).

Optionally, the modified relaxin may include one or more additionalsequence modifications, e.g., modifications that increase affinity foror selectivity of binding to a relaxin receptor. For example, themodified relaxin may include a modification at position 23 in the Achain, e.g., to a residue selected from alanine, glycine, serine,threonine, isoleucine, or leucine, which has been reported to increaseselectivity for activation of the relaxin receptor relative to wild-type(see U.S. 2011/0130332 which is hereby incorporated by reference).

In exemplary embodiments, the modified relaxin has one or morebiological activities of relaxin, such as activation of the relaxinreceptor RXFP1, e.g., human RXFP1. For example, said modified relaxinmay activate human RXFP1 with an EC50 less than 100 nM, less than 50 nM,less than 25 nM, than 10 nM, less than 5 nM, less than 4 nM, less than 3nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM,less than 0.1 nM, or lower. In further exemplary embodiments, saidmodified relaxin may activate human RXFP1 with an EC50 no more than1000-fold, 500-fold, 250-fold, 100-fold, 50-fold, 40-fold, 30-fold,20-fold, 10-fold, 5-fold, 2-fold greater than wild-type, or no greaterthan wild-type. Said EC50 may be determined using any of the methodsdescribed herein, e.g., in the examples, or any other method known inthe art.

Another embodiment provides a pharmaceutical composition or medicamentcomprising RLX-A-AQ1-20KPEG or another modified relaxin. A furtherembodiment provides the use of said modified relaxin in the treatment ofa cardiovascular disease or fibrotic disease, such as heart failure,pancreatitis, inflammation, cancer, scleroderma, pulmonary fibrosis,renal fibrosis, or hepatic fibrosis.

Compounds of the present disclosure may be used for reducing the area ofmyocardium affected by an infarction, and for the prophylaxis ofsecondary infarctions. Further provided is the use of the compounds ofthe present disclosure for the prophylaxis and/or treatment ofthromboembolic disorders, reperfusion damage following ischemia, micro-and macrovascular lesions (vasculitis), arterial and venous thromboses,edemas, ischemias such as myocardial infarction, stroke and transientischemic attacks, for cardio protection in connection with coronaryartery bypass operations (coronary artery bypass graft, CABG), primarypercutaneous transluminal coronary angioplasties (PTCAs), PTCAs afterthrombolysis, rescue PTCA, heart transplants and open-heart operations,and for organ protection in connection with transplants, bypassoperations, catheter examinations and other surgical procedures. Furtherprovided is the use of the compounds of the present disclosure for theprophylaxis and/or treatment of respiratory disorders, such as, forexample, chronic obstructive pulmonary disease (chronic bronchitis,COPD), interstitial lung disease, asthma, pulmonary emphysema,bronchiectases, cystic fibrosis (mucoviscidosis) and pulmonaryhypertension, in particular pulmonary arterial hypertension.

Exemplary embodiments of the present disclosure provide use ofRLX-A-AQ1-20KPEG or another modified relaxin as a medicament for theprophylaxis and/or treatment of kidney diseases, especially of acute andchronic kidney diseases and acute and chronic renal insufficiencies, aswell as acute and chronic renal failure, including acute and chronicstages of renal failure with and without the requirement of dialysis, aswell as the underlying or related kidney diseases such as renalhypoperfusion, dialysis induced hypotension, glomerulopathies,glomerular and tubular proteinuria, renal edema, hematuria, primary,secondary, as well as acute and chronic glomerulonephritis, membranousand membranoproliferative glomerulonephritis, Alport-Syndrome,glomerulosclerosis, interstistial tubular diseases, nephropathicdiseases, such as primary and inborn kidney diseases, renalinflammation, immunological renal diseases like renal transplantrejection, immune complex induced renal diseases, as well asintoxication induced nephropathic diseases, diabetic and non-diabeticrenal diseases, pyelonephritis, cystic kidneys, nephrosclerosis,hypertensive nephrosclerosis, nephrotic syndrome, that are characterizedand diagnostically associated with an abnormal reduction in creatinineclearance and/or water excretion, abnormal increased bloodconcentrations of urea, nitrogen, potassium and/or creatinine,alteration in the activity of renal enzymes, such as glutamylsynthetase,urine osmolarity and urine volume, increased microalbuminuria,macroalbuminuria, glomerular and arteriolar lesions, tubular dilation,hyperphosphatemia and/or the requirement of dialysis.

In addition, RLX-A-AQ1-20KPEG or another modified relaxin can be used asa medicament for the prophylaxis and/or treatment of renal carcinomas,after incomplete resection of the kidney, dehydration after overuse ofdiuretics, uncontrolled blood pressure increase with malignanthypertension, urinary tract obstruction and infection, amyloidosis, aswell as systemic diseases associated with glomerular damage, such asLupus erythematodes, and rheumatic immunological systemic diseases, aswell as renal artery stenosis, renal artery thrombosis, renal veinthrombosis, analgetics induced nephropathy and renal tubular acidosis.

In addition, the present disclosure provides the use of RLX-A-AQ1-20KPEGor another modified relaxin as a medicament for the prophylaxis and/ortreatment of contrast medium induced and drug induced acute and chronicinterstitial kidney diseases, metabolic syndrome and dyslipemia.

In addition, the present disclosure provides the use of RLX-A-AQ1-20KPEGor another modified relaxin as a medicament for the prophylaxis and/ortreatment of aftereffects associated with acute and/or chronic kidneydiseases, such as pulmonary edema, heart failure, uremia, anemia,electrolyte disturbances (e.g. hyperkalemia, hyponatremia), as well asbony and carbohydrate metabolism.

Further exemplary embodiments provide use of the RLX-A-AQ1-20KPEG orother modified relaxin for the treatment and/or prophylaxis of lungdiseases especially of asthmatic disorders, pulmonary arterialhypertension (PAH) and other forms of pulmonary hypertension (PH)including left-heart disease, HIV, sickle cell anemia, thromboembolisms(CTEPH), sarcoidosis, COPD-associated pulmonary hypertension, pulmonaryfibrosis-associated pulmonary hypertension, chronic-obstructivepulmonary disease (COPD), acute respiratory distress syndrome (ARDS),acute lung injury (ALI), alpha-1-antitrypsin deficiency (AATD),pulmonary fibrosis, pulmonary emphysema (for example pulmonary emphysemainduced by cigarette smoke) and cystic fibrosis (CF).

Further exemplary embodiments provide use of the RLX-A-AQ1-20KPEG orother modified relaxin for the treatment and/or prophylaxis of fibroticdisorders, including fibrotic disorders of the internal organs such as,for example, the lung, the heart, the kidney, the bone marrow (e.g.,myelofibrosis) and in particular the liver, and also dermatologicalfibroses and fibrotic eye disorders.

The present disclosure furthermore provides the use of RLX-A-AQ1-20KPEGor another modified relaxin for preparing a medicament for the treatmentand/or prevention of disorders, in particular the disorders mentionedabove.

The present disclosure furthermore provides a method for the treatmentand/or prevention of disorders, in particular the disorders mentionedabove, using an effective amount of at least one RLX-A-AQ1-20KPEG oranother modified relaxin.

The present disclosure furthermore provides RLX-A-AQ1-20KPEG or anothermodified relaxin for use in a method for the treatment and/orprophylaxis of coronary heart disease, acute coronary syndrome, heartfailure, and myocardial infarction.

The present disclosure also provides for pharmaceutical compositionscomprising RLX-A-AQ1-20KPEG or another modified relaxin in apharmacologically acceptable vehicle. The RLX-A-AQ1-20KPEG or othermodified relaxin may be administrated systemically or locally. Anyappropriate mode of administration known in the art may be usedincluding, but not limited to, intravenous, intraperitoneal,intraarterial, intranasal, transdermally, by inhalation, oral,subcutaneous administration, by local injection or in form of a surgicalimplant. For example, administration may be performed subcutaneously. Asa further example, administration may be performed intravenously.

The present disclosure also provides pharmaceutical compositions whichmay comprise RLX-A-AQ1-20KPEG or another modified relaxin, alone or incombination with at least one other agent, such as stabilizing compound,which may be administered in any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. In one embodiment, the pharmaceutically acceptablecarrier may be pharmaceutically inert. Any of these molecules can beadministered to a patient alone, or in combination with other agents,drugs or hormones, in pharmaceutical compositions where it is mixed withexcipient(s) or pharmaceutically acceptable carriers. In this context,the term combination encompasses any means of concurrent administration,whether or not the RLX-A-AQ1-20KPEG or other modified relaxin and theother agent are contained in the same composition or administeredseparately, which administration may be through the same or differentmodes of administration.

The present disclosure also provides methods comprising theadministration of pharmaceutical compositions disclosed herein. Suchadministration is accomplished orally or parenterally. Methods ofparenteral delivery include topical, transdermal, intra-arterial,intramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration. In addition to the active ingredients, thesepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Further details on techniques forformulation and administration may be found in the latest edition ofRemington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton,Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions may be formulated in aqueous solutions, for example inphysiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances that increase viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles may include fatty oils such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate or triglycerides, orliposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

RLX-A-AQ1-20KPEG or another modified relaxin can be used alone or incombination with other active compounds. The present disclosurefurthermore provides medicaments comprising at least oneRLX-A-AQ1-20KPEG or another modified relaxin and one or more furtheractive ingredients, in particular for the treatment and/or prevention ofthe disorders mentioned above.

Suitable active ingredients for combination may include, by way ofexample: active ingredients which modulate lipid metabolism,antidiabetics, hypotensive agents, perfusion-enhancing and/orantithrombotic agents, antioxidants, chemokine receptor antagonists,p38-kinase inhibitors, NPY agonists, orexin agonists, anorectics, PAF-AHinhibitors, antiphlogistics (COX inhibitors, LTB₄-receptor antagonists),analgesics for example aspirin, antidepressants and otherpsychopharmaceuticals.

The present disclosure further provides combinations of at least one ofRLX-A-AQ1-20KPEG or another modified relaxin with at least one lipidmetabolism-altering active ingredient, antidiabetic, blood pressurereducing active ingredient and/or agent having antithrombotic effects.

The RLX-A-AQ1-20KPEG or other modified relaxin can be combined with oneor more lipid metabolism-modulating active ingredients, by way ofexample from the group of the HMG-CoA reductase inhibitors, PCSK9antagonists, inhibitors of HMG-CoA reductase expression, squalenesynthesis inhibitors, ACAT inhibitors, LDL receptor inductors,cholesterol absorption inhibitors, polymeric bile acid adsorbers, bileacid reabsorption inhibitors, MTP inhibitors, lipase inhibitors, LpLactivators, fibrates, niacin, CETP inhibitors, PPAR-α, PPAR-γ and/orPPAR-δ agonists, RXR modulators, FXR modulators, LXR modulators, ApoAImimetics, thyroid hormones and/or thyroid mimetics, ATP citrate lyaseinhibitors, Lp(a) antagonists, cannabinoid receptor 1 antagonists,leptin receptor agonists, bombesin receptor agonists, histamine receptoragonists and the antioxidants/radical scavengers;

-   -   antidiabetics mentioned in the Rote Liste 2004/11, chapter 12,        and also, by way of example, those from the group of the        sulfonylurcas, biguanides, meglitinide derivatives, glucosidase        inhibitors, inhibitors of dipeptidyl-peptidase IV (DPP-IV        inhibitors), oxadiazolidinones, thiazolidinediones, GLP 1        receptor agonists, glucagon antagonists, insulin sensitizers,        CCK 1 receptor agonists, leptin receptor agonists, inhibitors of        liver enzymes

involved in the stimulation of gluconeogenesis and/or glycogenolysis,modulators of glucose uptake and also potassium channel openers, suchas, for example, those disclosed in WO 97/26265 and WO 99/03861;

-   -   hypotensive active ingredients, by way of example from the group        of the calcium antagonists, angiotensin All antagonists, ACE        inhibitors, renin inhibitors, beta-receptor blockers,        alpha-receptor blockers, aldosterone antagonists,        mineralocorticoid receptor antagonists, ECE inhibitors, ACE/NEP        inhibitors and the vasopeptidase inhibitors; and/or    -   antithrombotic agents, by way of example from the group of the        platelet aggregation inhibitors or the anticoagulants;    -   diuretics;    -   vasopressin receptor antagonists;    -   organic nitrates and NO donors;    -   compounds with positive inotropic activity;    -   compounds which inhibit the degradation of cyclic guanosine        monophosphate (cGMP) and/or cyclic adenosine monophosphate        (cAMP), such as, for example, inhibitors of phosphodiesterases        (PDE) 1, 2, 3, 4 and/or 5, in particular PDE 5 inhibitors, such        as sildenafil, vardenafil and tadalafil, and also PDE 3        inhibitors, such as milrinone;    -   natriuretic peptides, such as, for example, “atrial natriuretic        peptide” (ANP, anaritide), “B-type natriuretic peptide” or        “brain natriuretic peptide” (BNP, nesiritide). “C-type        natriuretic peptide” (CNP) and also urodilatin;    -   agonists of the prostacyclin receptor (IP receptor), such as, by        way of example, iloprost, beraprost, cicaprost;    -   inhibitors of the if (funny channel) channel, such as, by way of        example, ivabradine;    -   calcium sensitizers, such as, by way of example, levosimendan;    -   potassium supplements;    -   NO-independent, but heme-dependent stimulators of guanylate        cyclase, such as, in particular, the compounds described in WO        00/06568, WO 00/06569, WO 02/42301 and WO 03/095451;    -   NO- and heme-independent activators of guanylate cyclase, such        as, in particular, the

compounds described in WO 01/19355, WO 01/19776, WO 01/19778, WO01/19780, WO 02/070462 and WO 02/070510;

-   -   inhibitors of human neutrophil elastase (HNE), such as, for        example, sivelestat and DX-890 (Reltran);    -   compounds which inhibit the signal transduction cascade, such        as, for example, tyrosine-kinase inhibitors, in particular        sorafenib, imatinib, gefitinib and erlotinib; and/or    -   compounds which modulate the energy metabolism of the heart,        such as, for example, etomoxir, dichloroacetate, ranolazine and        trimetazidine.

Lipid metabolism-modifying active ingredients are to be understood asmeaning, for example, compounds from the group of the HMG-CoA reductaseinhibitors, squalene synthesis inhibitors, ACAT inhibitors, cholesterolabsorption inhibitors, MTP inhibitors, lipase inhibitors, thyroidhormones and/or thyroid mimetics, niacin receptor agonists, CETPinhibitors, PPAR-α agonists, PPAR-γ agonists, PPAR-δ agonists, polymericbile acid adsorbers, bile acid reabsorption inhibitors,antioxidants/radical scavengers and also the cannabinoid receptor 1antagonists.

In another embodiment, RLX-A-AQ1-20KPEG or another modified relaxin isadministered in combination with an HMG-CoA reductase inhibitor from theclass of the statins, such as, by way of example, lovastatin,simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin orpitavastatin.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a squalene synthesis inhibitor, suchas, by way of example, BMS-188494 or TAK-475.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with an ACAT inhibitor, such as, by way ofexample, avasimibe, melinamide, pactimibe, eflucimibe or SMP-797.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a cholesterol absorption inhibitor,such as, by way of example, ezetimibe, tiqueside or pamaqueside.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with an MTP inhibitor, such as, by way ofexample, implitapide, BMS-201038, R-103757 or JTT-130.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a lipase inhibitor, such as, by way ofexample, orlistat.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a thyroid hormone and/or thyroidmimetic, such as, by way of example, D-thyroxine or3,5,3′-triiodothyronine (T3).

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with an agonist of the niacin receptor, suchas, by way of example, niacin, acipimox, acifran or radecol.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a CETP inhibitor, such as, by way ofexample, dalcetrapib, BAY 60-5521, anacetrapib or CETP vaccine (CETi-1).

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a PPAR-γ agonist, for example from theclass of the thiazolidinediones, such as, by way of example,pioglitazone or rosiglitazone.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a PPAR-δ agonist, such as, by way ofexample, GW-501516 or BAY 68-5042.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a polymeric bile acid adsorber, suchas, by way of example, cholestyramine, colestipol, colesolvam,CholestaGel or colestimide.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a bile acid reabsorption inhibitor,such as, by way of example, ASBT (=IBAT) inhibitors, such as, forexample, AZD-7806, S-8921, AK-105, BARI-1741, SC-435 or SC-635.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with an antioxidant/radical scavenger, suchas, by way of example, probucol, AGI-1067, BO-653 or AEOL-10150.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a cannabinoid receptor 1 antagonist,such as, by way of example, rimonabant or SR-147778.

Antidiabetics are to be understood as meaning, for example, insulin andinsulin derivatives, and also orally effective hypoglycemic activeingredients. Here, insulin and insulin derivatives include both insulinsof animal, human or biotechnological origin and also mixtures thereof.The orally effective hypoglycemic active ingredients for example mayinclude sulfonylureas, biguanides, meglitinide derivatives, glucosidaseinhibitors and PPAR-gamma agonists.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with insulin.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a sulfonylurea, such as, by way ofexample, tolbutamide, glibenclamide, glimepiride, glipizide orgliclazide.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a biguanide, such as, by way ofexample, metformin.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a meglitinide derivative, such as, byway of example, rpaglinide or nateglinide.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a glucosidase inhibitor, such as, byway of example, miglitol or acarbose.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a DPP-IV inhibitor, such as, by way ofexample, sitagliptin and vildagliptin.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a PPAR-gamma agonist, for example fromthe class of the thiazolinediones, such as, by way of example,pioglitazone or rosiglitazone.

The hypotensive agents are for example understood as meaning compoundsfrom the group of the calcium antagonists, angiotensin All antagonists,ACE inhibitors, beta-receptor blockers, alpha-receptor blockers anddiuretics.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a calcium antagonist, such as, by wayof example, nifedipine, amlodipine, verapamil or diltiazem.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with an angiotensin All antagonist, such as,by way of example, losartan, valsartan, candesartan, embusartan,olmesartan or telmisartan.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with an ACE inhibitor, such as, by way ofexample, enalapril, captopril, lisinopril, ramipril, delapril,fosinopril, quinopril, perindopril or trandopril.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a beta-receptor blocker, such as, byway of example, propranolol, atenolol, timolol, pindolol, alprenolol,oxprenolol, penbutolol, bupranolol, metipranolol, nadolol, mepindolol,carazalol, sotalol, metoprolol, betaxolol, celiprolol, bisoprolol,carteolol, esmolol, labetalol, carvedilol, adaprolol, landiolol,nebivolol, epanolol or bucindolol.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with an alpha-receptor blocker, such as, byway of example, prazosin.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a diuretic, such as, by way of example,furosemide, bumetanide, torsemide, bendroflumethiazide, chlorothiazide,hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide,trichloromethiazide, chlorothalidone, indapamide, metolazone,quinethazone, acetazolamide, dichlorophenamide, methazolamide, glycerol,isosorbide, mannitol, amiloride or triamteren.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with an aldosterone or mineralocorticoidreceptor antagonist, such as, by way of example, spironolactone oreplerenone.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a vasopressin receptor antagonist, suchas, by way of example, conivaptan, tolvaptan, lixivaptan or SR-121463.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with an organic nitrate or NO donor, suchas, by way of example, sodium nitroprusside, nitroglycerol, isosorbidemononitrate, isosorbide dinitrate, molsidomin or SIN-1, or incombination with inhalative NO.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a positive-inotropic compound, such as,by way of example, cardiac glycosides (digoxin), beta-adrenergic anddopaminergic agonists, such as isoproterenol, adrenaline, noradrenaline,dopamine or dobutamine.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with antisympathotonics, such as reserpine,clonidine or alpha-methyldopa, or in combination with potassium channelagonists, such as minoxidil, diazoxide, dihydralazine or hydralazine, orwith substances which release nitrogen oxide, such as glycerol nitrateor sodium nitroprusside.

Antithrombotics are to be understood as meaning, for example, compoundsfrom the group of the platelet aggregation inhibitors or theanticoagulants.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a platelet aggregation inhibitor, suchas, by way of example, aspirin, clopidogrel, ticlopidine or dipyridamol.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a thrombin inhibitor, such as, by wayof example, ximelagatran, melagatran, dabigatran, bivalirudin orclexane.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a GPIIb/IIIa antagonist, such as, byway of example, tirofiban or abciximab.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a factor Xa inhibitor, such as, by wayof example, rivaroxaban (BAY 59-7939), DU-176b, apixaban, otamixaban,fidexaban, razaxaban, fondaparinux, idraparinux, PMD-3112, YM-150,KFA-1982, EMD-503982, MCM-17, MLN-1021, DX 9065a, DPC 906, JTV 803,SSR-126512 or SSR-128428.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with heparin or a low molecular weight (LMW)heparin derivative.

In another embodiment, the RLX-A-AQ1-20KPEG or other modified relaxin isadministered in combination with a vitamin K antagonist, such as, by wayof example, coumarin.

Other embodiments provide combinations comprising at least one of theRLX-A-AQ1-20KPEG or other modified relaxin and also one or more furtheractive ingredients selected from the group consisting of HMG-CoAreductase inhibitors (statins), diuretics, beta-receptor blockers,organic nitrates and NO donors, ACE inhibitors, angiotensin Allantagonists, aldosterone and mineralocorticoid receptor antagonists,vasopressin receptor antagonists, platelet aggregation inhibitors andanticoagulants, and also their use for the treatment and/or preventionof the disorders mentioned above.

The present disclosure furthermore provides medicaments comprising atleast one RLX-A-AQ1-20KPEG or other modified relaxin, usually togetherwith one or more inert nontoxic pharmaceutically suitable auxiliaries,and also their use for the purposes mentioned above.

Pharmaceutical compositions suitable for use in the methods andcompositions of the present disclosure include compositions wherein theactive ingredients are contained in an effective amount to achieve theintended purpose, e.g. heart failure. The determination of an effectivedose is well within the capability of those skilled in the art in viewof the present disclosure.

For any compound, the therapeutically effective dose can be estimatedinitially either in in vitro assays, e.g. RXFP1 receptor activation, exvivo in isolated perfused rat hearts, or in animal models, usually mice,rats, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans.

A therapeutically effective dose refers to that amount ofRLX-A-AQ1-20KPEG or another modified relaxin that ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures invitro or in experimental animals, e.g., ED50 (the dose therapeuticallyeffective in 50% of the population) and LD50 (the dose lethal to 50% ofthe population). The dose ratio between therapeutic and toxic effects isthe therapeutic index, and it can be expressed as the ratio, ED50/LD50.Exemplary pharmaceutical compositions exhibit large therapeutic indices.The data obtained from in vitro assays and animal studies are used informulating a range of dosage for human use. The dosage of suchcompounds lies for example within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, sensitivityof the patient, and the route of administration.

Normal dosage amounts may vary from 0.1 to 100,000 milligrams totaldose, depending upon the route of administration. Guidance as toparticular dosages and methods of delivery is provided in theliterature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. Thoseskilled in the art will employ different formulations forpolynucleotides than for proteins or their inhibitors. Similarly,delivery of polynucleotides or polypeptides will be specific toparticular cells, conditions, locations, etc.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises a relaxin A chain polypeptide and arelaxin B chain polypeptide, wherein said relaxin A chain polypeptidehas a sequence at least 90% identical to SEQ ID NO: 4 or SEQ ID NO:35,and said relaxin B chain polypeptide has a sequence at least 90%identical to SEQ ID NO: 5 or SEQ ID NO: 6; and (b) the non-naturallyencoded amino acid is linked to a linker, polymer, or biologicallyactive molecule.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises a relaxin A chain polypeptide and arelaxin B chain polypeptide, wherein said relaxin A chain polypeptidehas a sequence at least 90% identical to SEQ ID NO: 4, and said relaxinB chain polypeptide has a sequence at least 90% identical to SEQ ID NO:5 or SEQ ID NO: 6; and (b) the non-naturally encoded amino acid has thestructure:

wherein the R group is any substituent other than the side chain foundin alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, pyrrolysine, or selenocystiene.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises the relaxin A chain polypeptide of SEQID NO: 4 and the relaxin B chain polypeptide of SEQ ID NO: 5 or SEQ IDNO: 6 which relaxin A and B chain polypeptide sequences contain one ortwo amino acid substitutions selected from substitution with natural ornon-naturally encoded amino acids; and (b) said relaxin A or B chainpolypeptide is substituted with a non-naturally encoded amino acidlinked to a linker, polymer, or biologically active molecule.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises the relaxin A chain polypeptide of SEQID NO: 4 and the relaxin B chain polypeptide of SEQ ID NO: 5 or SEQ IDNO: 6 which relaxin A and B chain polypeptide sequences contain one ortwo amino acid substitutions selected from substitution with natural ornon-naturally encoded amino acids; and (b) said relaxin A or B chainpolypeptide is substituted with a non-naturally encoded amino acidhaving the structure:

wherein the R group is any substituent other than the side chain foundin alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, pyrrolysine, or selenocystiene.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises the relaxin A chain polypeptide of SEQID NO: 4 and the relaxin B chain polypeptide of SEQ ID NO: 5 or SEQ IDNO: 6 which relaxin A and B chain polypeptide sequences contain onenatural amino acid substitution and one non-naturally encoded amino acidsubstitution; and (b) the non-naturally encoded amino acid is linked toa linker, polymer, or biologically active molecule.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises the relaxin A chain polypeptide of SEQID NO: 4 and the relaxin B chain polypeptide of SEQ ID NO: 5 or SEQ IDNO: 6 which relaxin A and B chain polypeptide sequences contain onenatural amino acid substitution and one non-naturally encoded amino acidsubstitution; and (b) the non-naturally encoded amino acid has thestructure:

wherein the R group is any substituent other than the side chain foundin alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, pyrrolysine, or selenocystiene.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises a relaxin A chain polypeptide and arelaxin B chain polypeptide, wherein said relaxin A chain polypeptidehas a sequence at least 80%, at least 85%, at least 90%, or at least 95%identical to SEQ ID NO: 4 or SEQ ID NO:35, and said relaxin B chainpolypeptide has a sequence at least 80%, at least 85%, at least 90%, orat least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 6; and (b) thenon-naturally encoded amino acid is linked to a linker, polymer, orbiologically active molecule.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises a relaxin A chain polypeptide and arelaxin B chain polypeptide, wherein said relaxin A chain polypeptidehas a sequence at least 80%, at least 85%, at least 90%, or at least 95%identical to SEQ ID NO: 4, and said relaxin B chain polypeptide has asequence at least 80%, at least 85%, at least 90%, or at least 95%identical to SEQ ID NO: 5 or SEQ ID NO: 6; and (b) the non-naturallyencoded amino acid has the structure:

wherein the R group is any substituent other than the side chain foundin alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, pyrrolysine, or selenocystiene.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises the relaxin A chain polypeptide of SEQID NO: 4 and the relaxin B chain polypeptide of SEQ ID NO: 5 or SEQ IDNO: 6 which relaxin A and B chain polypeptide sequences each contain upto one, two, three, or four amino acid substitutions selected fromsubstitution with natural or non-naturally encoded amino acids; and (b)said relaxin A or B chain polypeptide is substituted with anon-naturally encoded amino acid linked to a linker, polymer, orbiologically active molecule.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein: (a)the relaxin polypeptide comprises the relaxin A chain polypeptide of SEQID NO: 4 and the relaxin B chain polypeptide of SEQ ID NO: 5 or SEQ IDNO: 6 which relaxin A and B chain polypeptide sequences each contain upto one, two, three, or four amino acid substitutions selected fromsubstitution with natural or non-naturally encoded amino acids; and (b)said relaxin A or B chain polypeptide is substituted with anon-naturally encoded amino acid having the structure:

wherein the R group is any substituent other than the side chain foundin alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, theonine, tryptophan,tyrosine, valine, pyrrolysine, or selenocystiene.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinA chain having at least 80% identity to the polypeptide of SEQ ID NO:4.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinA chain having at least 85% identity to the polypeptide of SEQ ID NO:4.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinA chain having at least 90% identity to the polypeptide of SEQ ID NO:4.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinA chain having at least 95% identity to the polypeptide of SEQ ID NO:4.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having at least 80% identity to the polypeptide of SEQ ID NO:5.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having at least 85% identity to the polypeptide of SEQ ID NO:5.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having at least 90% identity to the polypeptide of SEQ ID NO:5.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having at least 95% identity to the polypeptide of SEQ ID NO:5.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having at least 80% identity to the polypeptide of SEQ ID NO:6.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having at least 85% identity to the polypeptide of SEQ ID NO:6.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having at least 90% identity to the polypeptide of SEQ ID NO:6.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having at least 95% identity to the polypeptide of SEQ ID NO:6.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinA chain having the polypeptide sequence of SEQ ID NO:4 with up to foursubstitutions.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinA chain having the polypeptide sequence of SEQ ID NO:4 with up to threesubstitutions.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinA chain having the polypeptide sequence of SEQ ID NO:4 with up to twosubstitutions.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinA chain having the polypeptide sequence of SEQ ID NO:4 with up to onesubstitution.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having the polypeptide sequence of SEQ ID NO:5 with up to foursubstitutions.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having the polypeptide sequence of SEQ ID NO:5 with up to threesubstitutions.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having the polypeptide sequence of SEQ ID NO:5 with up to twosubstitutions.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having the polypeptide sequence of SEQ ID NO:6 with up to onesubstitution.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having the polypeptide sequence of SEQ ID NO:6 with up to foursubstitutions.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having the polypeptide sequence of SEQ ID NO:6 with up to threesubstitutions.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having the polypeptide sequence of SEQ ID NO:6 with up to twosubstitutions.

In exemplary embodiments, the relaxin polypeptide may comprise a relaxinB chain having the polypeptide sequence of SEQ ID NO:6 with up to onesubstitution.

Said substitutions or non-identical amino acids may include natural ornon-naturally occurring amino acids. In exemplary embodiments saidrelaxin A and B chains are linked by one or more disulfide bonds,preferably by one or two of the interchain disulfide bonds of wild-typerelaxin as depicted in FIG. 4 . In exemplary embodiments, said relaxin Achain includes the intrachain disulfide bond of wild-type relaxin, asdepicted in FIG. 4 . Preferably the relaxin is biologically active. Inexemplary embodiments the relaxin binds to a relaxin receptor, such asthe human relaxin receptor RXFP1, such as with an EC50 of at most100-fold, 50-fold, 30-fold, or 10-fold greater than wild-type humanrelaxin 2.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein:

-   -   (a) the relaxin polypeptide comprises a relaxin A chain        polypeptide and a relaxin B chain polypeptide, wherein said        relaxin A chain polypeptide may have a sequence at least 80%        identical to SEQ ID NO: 4, and said relaxin B chain polypeptide        has a sequence at least 80% identical to SEQ ID NO: 5 or SEQ ID        NO: 6; and    -   (b) the non-naturally encoded amino acid is linked to a linker,        polymer, or biologically active molecule.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein:

-   -   (a) the relaxin polypeptide comprises a relaxin A chain        polypeptide and a relaxin B chain polypeptide, wherein said        relaxin A chain polypeptide has a sequence at least 80%        identical to SEQ ID NO: 4, and said relaxin B chain polypeptide        has a sequence at least 80% identical to SEQ ID NO: 5 or SEQ ID        NO: 6; and    -   (b) the non-naturally encoded amino acid has the structure:

wherein the R group is any substituent other than the side chain foundin alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, pyrrolysine, or selenocystiene.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein:

-   -   (a) the relaxin polypeptide comprises the relaxin A chain        polypeptide of SEQ ID NO: 4 and the relaxin B chain polypeptide        of SEQ ID NO: 5 or SEQ ID NO: 6 which relaxin A chain        polypeptide sequence contains up to four amino acid        substitutions selected from natural or non-naturally encoded        amino acids, which relaxin B chain polypeptide sequences contain        up to four amino acid substitutions selected from substitution        with natural or non-naturally encoded amino acids; and    -   (b) said relaxin A or B chain polypeptide is substituted with a        non-naturally encoded amino acid linked to a linker, polymer, or        biologically active molecule.

In another aspect, the disclosure provides a modified relaxinpolypeptide comprising a non-naturally encoded amino acid, wherein:

-   -   (a) the relaxin polypeptide comprises the relaxin A chain        polypeptide of SEQ ID NO: 4 and the relaxin B chain polypeptide        of SEQ ID NO: 5 or SEQ ID NO: 6 which relaxin A chain        polypeptide sequence contains up to four amino acid        substitutions selected from natural or non-naturally encoded        amino acids, which relaxin B chain polypeptide sequences contain        up to four amino acid substitutions selected from substitution        with natural or non-naturally encoded amino acids; and    -   (b) said relaxin A or B chain polypeptide is substituted with a        non-naturally encoded amino acid having the structure:

wherein the R group is an substituent other than the side chain found inalanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, valine, pyrrolysine, or selenocystiene.

Said relaxin A chain polypeptide may have a sequence at least 85%, atleast 90%, or at least 95% identical to SEQ ID NO: 4. Said relaxin Bchain polypeptide may have a sequence at least 85%, at least 90%, or atleast 95% identical to SEQ ID NO: 5 or SEQ ID NO:6. Said relaxin A chainmay comprise the polypeptide of SEQ ID NO: 4 containing up to three, upto two, up to one, or zero amino acid substitutions. Said relaxin Bchain may comprise the polypeptide of SEQ ID NO: 5 or SEQ ID NO: 6containing up to three, up to two, up to one, or zero amino acidsubstitutions.

Said non-naturally encoded amino acid may be a phenylalanine analog orderivative. The non-naturally encoded amino acid may have the structure:

wherein the R group may be any substituent other than the side chainfound in alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine, pyrrolysine, or selenocystiene and may besubstituted at a position selected from the group consisting of residues1, 2, 5, and 18 of SEQ ID NO: 4, residues 5, 7, 18, and 28 of SEQ ID NO:5, and residues 5, 7, 18, and 28 of SEQ ID NO: 6.

The non-naturally encoded amino acid may be linked to a linker, polymer,or biologically active molecule, the non-naturally encoded amino acidmay comprise a first functional group, and the linker, polymer, orbiologically active molecule may comprise a second functional group,wherein the first functional group and second functional group are notidentical and each comprise a carbonyl group, an aminooxy group, ahydrazide group, a hydrazine group, a semicarbazide group, an azidegroup, or an alkyne group.

The first functional group on the non-naturally encoded amino acid maybe a carbonyl moiety and the second functional group contained on thelinker, polymer, or biologically active molecule may be an aminooxymoiety, and the resultant covalent linkage created by the reaction ofthe first and second functional groups may be an oxime linkage.Alternatively, the first functional group on the non-naturally encodedamino acid may be an azide moiety and the second functional groupcontained on the linker, polymer, or biologically active molecule may bean alkyne moiety.

Said modified relaxin polypeptide may be recombinant, may be produced ina eukaryotic host cell, may be produced in a non-eukaryotic host cell,or may be produced by in vitro translation.

The non-naturally encoded amino acid may be linked to a polymer. Thepolymer may comprise polyethylene glycol. The polyethylene glycol mayhave an average molecular weight of between about 0.1 kDa and about 100kDa. The polyethylene glycol may have an average molecular weight ofabout 20 kDa.

The non-naturally encoded amino acid may be substituted at a positionselected from the group consisting of residues 1, 2, 5, and 18 of SEQ IDNO: 4, residues 5, 7, 18, and 28 of SEQ ID NO: 5, and residues 5, 7, 18,and 28 of SEQ ID NO: 6.

The modified relaxin polypeptide may be therapeutically effective todecrease vasoconstriction, increase nitric oxide (NO) production,decrease platelet aggregation, decrease hypertrophy, decreaseCF-stimulated protein synthesis, treat angiotensin-II (AngII)-mediatedvasoconstriction, treat endothelin-1 (ET-1)-mediated vasoconstriction,or increase Atrial natriuretic peptide (ANP) expression, or retains theeffect of the wild-type relaxin polypeptide of SEQ ID NO: 1 on urineoutput, plasma BUN, ad libitum water intake, plasma sodium, or plasmaosmolarity.

The modified relaxin polypeptide may exhibit an increased in vivohalf-life relative to the human relaxin polypeptide of SEQ ID NO: 1. Thehalf-life may be increased by an amount selected from at least 2-fold3-fold, 4-fold, 5-fold or 10-fold.

The non-naturally encoded amino acid may be substituted at residue 1 inthe relaxin A chain polypeptide.

The non-naturally encoded amino acid may be selected from apara-substituted, ortho-substituted, or meta-substituted phenylalaninecomprising a carbonyl group, an aminooxy group, a hydrazide group, ahydrazine group, a semicarbazide group, an azide group, or an alkynegroup. The non-naturally encoded amino acid may bepara-acetyl-L-phenylalanine.

In another aspect, the disclosure provides a composition which maycomprise a modified relaxin polypeptide as described herein and apharmaceutically acceptable carrier. The composition may be formulatedfor a mode of administration selected from the group consisting ofsubcutaneous administration and intravenous administration.

In another aspect the disclosure provides a composition which maycomprise a modified relaxin polypeptide which may contain at least onenon-naturally encoded amino acid. Said composition may be formulated fora mode of administration selected from the group consisting ofsubcutaneous administration and intravenous administration. Saidmodified relaxin polypeptide may comprise an A chain and a B chainlinked by at least one disulfide bond, wherein said A chain may compriseSEQ ID NO: 4 substituted with up to one, two, three, or four natural ornon-naturally encoded amino acids, and said B chain may comprise SEQ IDNO: 6 substituted with up to one, two, three, or four natural ornon-naturally encoded amino acids. Said A chain may comprise SEQ ID NO:4 substituted with up to one or two natural or non-naturally encodedamino acids. Said B chain may comprise SEQ ID NO: 6 substituted with upto one or two natural or non-naturally encoded amino acids or whereinsaid B chain may comprise SEQ ID NO: 6. Said A chain may be substitutedwith a non-naturally encoded amino acid at position 1. The non-naturallyencoded amino acid at position 1 of the A chain may be linked to a watersoluble polymer. Said modified relaxin polypeptide may be linked to awater soluble polymer. Said water soluble polymer may comprise apolyethylene glycol (PEG). The molecular weight of said PEG may bebetween about 2 kDA and about 40 kDA, or between about 17 kDA and 23kDA. Said mode of administration may be subcutaneous administration.

The composition as herein described may further comprise one or moreother active compounds selected from: antidiabetics, hypotensive agents,perfusion-enhancing agents, lipid metabolism modulators, antithromboticagents, antioxidants, chemokine receptor antagonists, p38-kinaseinhibitors, NPY agonists, orexin agonists, anorectics, PAF-AHinhibitors, antiphlogistics, COX inhibitors, LTB₄-receptor antagonists,analgesics, and aspirin.

In another aspect, the disclosure provides a method of treatment ofdisease, which may comprise administering a modified relaxin polypeptideas herein described or a composition containing a modified relaxinpolypeptide as herein described to a subject in need thereof.

In another aspect, the disclosure provides a method of treatment ofdisease, which may comprise administering a modified relaxin polypeptideto a subject in need thereof. Said modified relaxin polypeptide maycomprise an A chain and a B chain linked by at least one disulfide bond,wherein said A chain may comprise SEQ ID NO: 4 substituted with up totwo natural or non-naturally encoded amino acids, and said B chain maycomprise SEQ ID NO: 6 substituted with up to two natural ornon-naturally encoded amino acids. Said A chain may comprise SEQ ID NO:4 substituted with up to one natural or non-naturally encoded aminoacid. Said B chain may comprise SEQ ID NO: 6 substituted with up to onenatural or non-naturally encoded amino acid or wherein said B chain maycomprise SEQ ID NO: 6. Said A chain may be substituted with anon-naturally encoded amino acid at position 1. The non-naturallyencoded amino acid at position 1 of the A chain may be linked to a watersoluble polymer. Said modified relaxin polypeptide may be linked to awater soluble polymer. Said water soluble polymer may comprise apolyethylene glycol (PEG). The molecular weight of said PEG may bebetween about 2 kDA and about 40 kDA, or between about 17 kDA and 23kDA. The disease may comprise heart failure. Said heart failure maycomprise one or more of acute decompensated heart failure, right heartfailure, left heart failure, global failure, ischemic cardiomyopathy,dilated cardiomyopathy, heart failure associated with congenital heartdefects, heart failure associated with heart valve defects, mitralstenosis, mitral insufficiency, aortic stenosis, aortic insufficiency,tricuspid stenosis, tricuspid insufficiency, pulmonary stenosis,pulmonary valve insufficiency, heart failure associated with combinedheart valve defects, myocardial inflammation (myocarditis), chronicmyocarditis, acute myocarditis, viral myocarditis, diabetic heartfailure, alcoholic cardiomyopathy, heart failure associated with cardiacstorage disorders, diastolic heart failure, and systolic heart failure.

The disease may comprise cardiovascular disease, lung disease, fibroticdisease or kidney disease. The disease may comprise pancreatitis,inflammation, cancer, scleroderma, pulmonary fibrosis, renal fibrosis,or hepatic fibrosis. The disease may comprise fibrosis of the lung,heart, kidney, bone marrow, liver, dermatological fibrosis, or afibrotic eye disorder. The disease may comprise one or more of ischemia,Alzheimer's disease, corneal injury, neurodegenerative disease,cardiovascular disease, fibrotic disease, failure, pancreatitis,inflammation, cancer, scleroderma, pulmonary fibrosis, renal fibrosis,hepatic fibrosis, thromboembolic disorders, reperfusion damage followingischemia, micro- and macrovascular lesions (vasculitis), arterial andvenous thromboses, edemas, ischemias, myocardial infarction, stroke,transient ischemic attack, cardio protection in connection with coronaryartery bypass operations, cardio protection in connection with primarypercutaneous transluminal coronary angioplasties (PTCAs), PTCAs afterthrombolysis, rescue PTCA, heart transplants and open-heart operations,organ protection in connection with transplants, bypass operations,catheter examinations and other surgical procedures, respiratorydisorders, chronic obstructive pulmonary disease, chronic bronchitis,interstitial lung disease, asthma, pulmonary emphysema, bronchiectases,cystic fibrosis (mucoviscidosis) and pulmonary hypertension, inparticular pulmonary arterial hypertension, kidney disease, acute andchronic kidney diseases and acute and chronic renal insufficiencies, aswell as acute and chronic renal failure with or without the requirementof dialysis, underlying or related kidney diseases, renal hypoperfusion,dialysis induced hypotension, glomerulopathies, glomerular and tubularproteinuria, renal edema, hematuria, chronic glomerulonephritis(including primary, secondary, or acute), membranous andmembranoproliferative glomerulonephritis, Alport-Syndrome,glomerulosclerosis, interstistial tubular diseases, nephropathicdiseases, primary and inborn kidney diseases, renal inflammation,immunological renal diseases, renal transplant rejection, immune complexinduced renal diseases, intoxication induced nephropathic diseases,diabetic and non-diabetic renal diseases, pyelonephritis, cystickidneys, nephrosclerosis, hypertensive nephrosclerosis, nephroticsyndrome, diseases that are characterized and diagnostically associatedwith an abnormal reduction in creatinine clearance and/or waterexcretion, abnormal increased blood concentrations of urea, nitrogen,potassium and/or creatinine, alteration in the activity of renal enzymesincluding without limitation glutamylsynthetase, urine osmolarity andurine volume, increased microalbuminuria, macroalbuminuria, glomerularand arteriolar lesions, tubular dilation, hyperphosphatemia, diseaserequiring dialysis for treatment, renal carcinomas, after incompleteresection of the kidney, dehydration after overuse of diuretics,uncontrolled blood pressure increase with malignant hypertension,urinary tract obstruction and infection, amyloidosis, systemic diseasesassociated with glomerular damage, Lupus erythematodes, rheumaticimmunological systemic diseases, renal artery stenosis, renal arterythrombosis, renal vein thrombosis, analgetics induced nephropathy,polycystic kidney disease, renal tubular acidosis, contrast mediuminduced and drug induced acute and chronic interstitial kidney diseases,metabolic syndrome, dyslipemia, aftereffects associated with acuteand/or chronic kidney diseases, pulmonary edema, heart failure, uremia,anemia, electrolyte disturbances, hyperkalemia, hyponatremia, bony andcarbohydrate metabolism, lung diseases, asthmatic disorders, pulmonaryarterial hypertension (PAH), pulmonary hypertension (PH), left-heartdisease, HIV, sickle cell anemia, thromboembolisms (CTEPH), sarcoidosis,COPD-associated pulmonary hypertension, pulmonary fibrosis-associatedpulmonary hypertension, chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), acute lung injury (ALI),alpha-1-antitrypsin deficiency (AATD), pulmonary fibrosis, pulmonaryemphysema, pulmonary emphysema induced by cigarette smoke, cysticfibrosis (CF), fibrotic disorders, fibrotic disorders of the internalorgans, fibrotic disorders of the lung, fibrotic disorders of the heart,fibrotic disorders of the kidney, fibrotic disorders of the bone marrowfibrotic disorders of the liver, dermatological fibroses, fibrotic eyedisorders, osteodegenerative joint dysfunction, angiotensin-II(AngII)-mediated vasoconstriction, endothelin-1 (ET-1)-mediatedvasoconstriction, ischemic conditions, ischemia associated withmyocardial infarct ischemia associated with wounds, renal pathologies,renal pathologies related to vasoconstriction, or hypertension.

In another aspect, the disclosure provides a modified relaxinpolypeptide as herein described or a composition containing a modifiedrelaxin polypeptide as herein described for use as a medicament.

In another aspect, the disclosure provides the use of a modified relaxinpolypeptide as herein described or the use of a composition containing amodified relaxin polypeptide as herein described for the manufacture ofa medicament for treating heart failure, cardiovascular disease, lungdisease, fibrotic disease, kidney disease, pancreatitis, inflammation,cancer, scleroderma, pulmonary fibrosis, renal fibrosis, hepaticfibrosis, coronary fibrosis, bone marrow fibrosis, dermatologicalfibrosis, a fibrotic eye disorder, ischemia, Alzheimer's disease, or acorneal injury.

In another aspect, the disclosure provides a modified relaxinpolypeptide as herein described or composition containing a modifiedrelaxin polypeptide as herein described, wherein said modified relaxinpolypeptide may be conjugated to at least one substance including butnot limited to a label, a dye, a polymer, a water-soluble polymer, aderivative of polyethylene glycol, a photocrosslinker, a radionuclide, acytotoxic compound, a drug, an affinity label, a photoaffinity label, areactive compound, a resin, another polypeptide or protein, apolypeptide analog, an antibody, an antibody fragment, a metal chelator,a cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, aRNA, an antisense polynucleotide, a saccharide, a water-solubledendrimer, a cyclodextrin, an inhibitory ribonucleic acid, abiomaterial, a nanoparticle, a spin label, a fluorophore, ametal-containing moiety, a radioactive moiety, a functional group, agroup that covalently or noncovalently interacts with other molecules, aphotocaged moiety, an actinic radiation excitable moiety, aphotoisomerizable moiety, biotin, a derivative of biotin, a biotinanalogue, a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, an elongated side chain, a carbon-linkedsugar, a redox-active agent, an amino thioacid, a toxic moiety, anisotopically labeled moiety, a biophysical probe, a phosphorescentgroup, a chemiluminescent group, an electron dense group, a magneticgroup, an intercalating group, a chromophore, an energy transfer agent,a biologically active agent, a detectable label, a small molecule, aquantum dot, a nanotransmitter, a radionucleotide, a radiotransmitter, aneutron-capture agent, or any combination of the above.

In yet another aspect, the invention provides relaxin polypeptidesoptionally modified with at least one non-naturally encoded amino acid.This specification will provide some embodiments, however it should beappreciated that these embodiments are for the purpose of illustratingthe invention, and are not to be construed as limiting the scope of theinvention as defined by the claims.

In another aspect of the present invention, relaxin polypeptides with atleast one non-naturally encoded amino acid are attached to at least onewater soluble polymer.

In one aspect, the invention relates to a method of promotingangiogenesis in a mammal in need thereof by administering atherapeutically effective amount of relaxin. In another embodiment,relaxin is administered in an amount sufficient to maintain a serumconcentration of at least about 1 ng/ml. In a further embodiment therelaxin polypeptide is human relaxin (hR2).

The present invention provides methods of treating individuals withdiminished arterial compliance an effective amount of a formulationcomprising a relaxin receptor agonist. In a preferred embodiment therelaxin receptor agonist is a recombinant human relaxin, e.g., human H2relaxin.

In one embodiment of the invention, the invention provides a method ofincreasing arterial compliance in a subject, wherein said methodcomprises measuring global arterial compliance in said subject:determining that said global arterial compliance is diminished in saidsubject relative to global arterial compliance in a healthy subject; andadministering to said subject a pharmaceutical formulation comprisingrelaxin to increase arterial compliance in said subject. Global arterialcompliance may be measured, in one embodiment, from the diastolic decayof the aortic pressure waveform using the area method. In anotherembodiment, global arterial compliance may be calculated as the strokevolume-to-pulse pressure ratio, where the stroke volume is defined asthe ratio of cardiac output to heart rate.

In related embodiments, the local arterial compliance or the regionalarterial compliance of a subject may be measured in addition to or as analternative to the global arterial compliance measurement and, if thelocal or regional arterial compliance is diminished relative to thelocal or regional arterial compliance expected for a similarly situatedhealthy individual, relaxin may be administered to increase arterialcompliance in that individual.

In further embodiments, the subject to whom relaxin is administeredsuffers from one or more of the following disorders: atherosclerosis,Type 1 diabetes, Type 2 diabetes, coronary artery disease, scleroderma,stroke, diastolic dysfunction, familial hypercholesterolemia, isolatedsystolic hypertension, primary hypertension, secondary hypertension,left ventricular hypertrophy, arterial stiffness associated withlong-term tobacco smoking, arterial stiffness associated with obesity,arterial stiffness associated with age, systemic lupus erythematosus,preeclampsia, and hypercholesterolemia. In related embodiments, theinvention provides methods of increasing arterial compliance inperimenopausal, menopausal, and post-menopausal women and in individualswho are at risk of one of the aforementioned disorders.

In an additional embodiment of the invention, administration of relaxinincreases arterial compliance by at least 10%, 15%, 20% or more,relative to the measured arterial compliance before administration. Instill further embodiments, the invention provide for the administrationof relaxin to individuals with diminished arterial compliance at apredetermined rate so as to maintain a serum concentration of relaxinfrom 0.5 to 80 ng/ml. In one embodiment, the relaxin is recombinanthuman relaxin with one non-naturally encoded amino acid. In yet anotherembodiment, the relaxin is relaxin with more than one non-naturallyencoded amino acid. In yet another embodiment of the present invention,the relaxin has a non-naturally encoded amino acid linked to a watersoluble polymer. In related embodiments, the relaxin may be administereddaily, in an injectable formulation, as a sustained release formulation,or as a continuous infusion.

In another aspect, the invention relates to the treatment of infectionsor ischemic wounds by administering a therapeutically effective amountof relaxin. In a particularly preferred embodiment, the infection orischemic wound is one where injury has resulted from lack of oxygen dueto poor circulation.

In yet another aspect of the invention, there is provided a method ofusing relaxin polypeptides of the present invention for the manufactureof a medicament for the treatment of an infection or ischemic wound, orfor the manufacture of a medicament for the promotion of angiogenesis.In another aspect, the present invention relates to the treatment ofosteodegenerative joint dysfunction, and in another aspect the treatmentof the osteodegenerative joint dysfunction comprises hR2 in addition toone or more adjuvants, including but not limited to glucosamine. Inanother aspect, the present invention relates to the treatment ofAlzheimer's disease, and in another aspect the treatment of theAlzheimer's disease comprises hR2 in addition to one or more adjuvants,including but not limited to estrogen. In another embodiment, thisinvention relates to a method of modulating the reproductive physiologyof mammals comprising administering to the mammal a therapeuticallyeffective amount of the composition herein.

The invention further provides methods for treating angiotensin-II(AngII)-mediated vasoconstriction. These methods generally compriseadministering a formulation comprising an amount of relaxin effective toreverse, inhibit, or reduce the vasoconstricting effects of AngII.

The invention further provides methods for treating endothelin-1(ET-1)-mediated vasoconstriction. These methods generally compriseadministering a formulation comprising an amount of relaxin effective toreverse, inhibit, or reduce the vasoconstricting effects of ET-1. Insome embodiments, the methods comprise increasing endothelin type Breceptor activation in a cell in a blood vessel by administering relaxinto the individual.

The invention further provides methods for treating an ischemiccondition, generally comprising administering a formulation comprisingan amount of relaxin effective to stimulate or promote angiogenesisand/or vasodilation, thereby treating the ischemic condition. Themethods are useful in treating a variety of ischemic conditions. In someembodiments, methods are provided for treating an ischemic conditionwhich arises as a result of myocardial infarct. In other embodiments,methods are provided for treating an ischemic condition associated witha wound. Thus, the invention further provides methods for promotingwound healing.

The invention further provides methods for stimulating angiogenic and/orvasodilatory cytokine expression generally comprising administering aformulation comprising an amount of relaxin effective to vasodilateblood vessels and/or stimulate or promote angiogenic cytokineproduction. In some embodiments, the methods provide for stimulatingexpression of basic fibroblast growth factor (bFGF) and/or vascularendothelial cell growth factor (VEGF). Such methods are useful intreating a wide variety of diseases which can be treated by increasingblood flow at or near the site of disease.

The invention further provides a method of increasing renal vasodilationand hyperfiltration, generally comprising administering a formulationcomprising an amount of relaxin. These methods are useful in treating avariety of renal pathologies. Accordingly, the invention furtherprovides methods of treating a renal pathology related tovasoconstriction.

The invention further provides a method of reducing pulmonaryhypertension, generally comprising administering a formulationcomprising an amount of relaxin.

In a patents assigned to Connetics Corporation and to BAS Medical, Inc.,U.S. Pat. Nos. 6,211,147 and 6,780,836 respectively, both incorporatedherein by reference, methods of promoting angiogenesis using relaxinwere disclosed. In a patent assigned to Genentech, Inc., U.S. Pat. No.5,759,807, which is herein incorporated by reference, a process forprokaryotic production of relaxin from prorelaxin is disclosed. Yue U.S.Pat. No. 6,251,863 discloses methods of treating osteodegenerative jointdysfunction and methods of treating Alzheimer's by administering relaxinmedicaments further comprising glucosamine sulfate and estrogen,respective for each of the conditions, and the specification of thispatent is also herein incorporated by reference in its entirety.

In some embodiments, the relaxin polypeptide comprises one or morepost-translational modifications. In some embodiments, the relaxinpolypeptide is linked to a linker, polymer, or biologically activemolecule. In some embodiments, the relaxin polypeptide is linked to abifunctional polymer, bifunctional linker, or at least one additionalrelaxin polypeptide.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thenon-naturally encoded amino acid is linked to the water soluble polymerwith a linker or is bonded to the water soluble polymer. In someembodiments, the poly(ethylene glycol) molecule is a bifunctionalpolymer. In some embodiments, the bifunctional polymer is linked to asecond polypeptide. In some embodiments, the second polypeptide is arelaxin polypeptide.

In some embodiments, the relaxin polypeptide comprises at least twoamino acids linked to a water soluble polymer comprising a poly(ethyleneglycol) moiety. In some embodiments, at least one amino acid is anon-naturally encoded amino acid.

In some embodiments, the relaxin polypeptide comprises at least twoamino acids linked to a water soluble polymer comprising a poly(ethyleneglycol) moiety. In some embodiments, at least one amino acid is anon-naturally encoded amino acid.

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of therelaxin or prorelaxin polypeptides, relaxin analogs, prorelaxin, relaxinA chain, relaxin B chain Ala1Asp relaxin, or relaxin polypeptides: inthe A chain before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25 (i.e., at the carboxyl terminus of the protein), and any combinationthereof (SEQ ID NO: 4) and/or in the B chain before position 1 (i.e. atthe N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 (SEQ ID NO: 5 orthe corresponding amino acids in SEQ ID NO: 6). In some embodiments, oneor more non-naturally encoded amino acids are incorporated in one ormore of the following positions in any of the relaxin or prorelaxinpolypeptides: in the A chain before position 1 (i.e. at the N-terminus),1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 (i.e., atthe carboxyl terminus of the protein), and any combination thereof (SEQID NO: 1 or the corresponding amino acids in SEQ ID NO: 2 or 3). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in relaxin:before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89 (i.e., at the carboxyl terminus of theprotein of SEQ ID NO: 3).

In some embodiments a non-naturally encoded amino acid is incorporatedin the A chain at amino acid position 1 (SEQ ID NO: 4). In someembodiments a non-naturally encoded amino acid is incorporated in the Achain at amino acid position 5 (SEQ ID NO: 4). In some embodiments anon-naturally encoded amino acid is incorporated in the B chain at aminoacid position 7 (SEQ ID NO: 5 or SEQ ID NO: 6). In some embodiments anon-naturally encoded amino acid is incorporated in the A chain at aminoacid position 2 (SEQ ID NO: 4). In some embodiments a non-naturallyencoded amino acid is incorporated in the A chain at amino acid position13 (SEQ ID NO: 4). In some embodiments a non-naturally encoded aminoacid is incorporated in the B chain at amino acid position 5 (SEQ ID NO:5 or SEQ ID NO: 6). In some embodiments a non-naturally encoded aminoacid is incorporated in the A chain at amino acid position 18. In someembodiments a non-naturally encoded amino acid is incorporated in the Bchain at amino acid position 5. In some embodiments a non-naturallyencoded amino acid is incorporated in the B chain at amino acid position28. In some embodiments, one or more non-naturally encoded amino acidsare incorporated in one of the following positions in the relaxinpolypeptides: in the A chain at amino acid position 1, 2, 5, 13, 18 (SEQID NO: 4 or the corresponding amino acids in SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, or other known relaxin sequences). In some embodiments,one or more non-naturally encoded amino acids are incorporated in one ofthe following positions in the relaxin polypeptides: in the A chain atamino acid position 1, 2, 5, 13 (SEQ ID NO: 4 or the corresponding aminoacids in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or other knownrelaxin sequences). In some embodiments, one or more non-naturallyencoded amino acids are incorporated in one of the following positionsin the relaxin polypeptides: in the A chain at amino acid position 1, 2,5 (SEQ ID NO: 4 or the corresponding amino acids in SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, or other known relaxin sequences). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one of the following positions in the relaxinpolypeptides: in the A chain at amino acid position 2 or 5 (SEQ ID NO: 4or the corresponding amino acids in SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, or other known relaxin sequences).

In some embodiments, a non-naturally encoded amino acids is incorporatedin one of the following positions in relaxin polypeptides: in the Bchain at amino acid position 5 or 7 (SEQ ID NO: 5 or SEQ ID NO: 6, orthe corresponding amino acid positions in SEQ ID NOs: 1, 2, or 3). Insome embodiments, a non-naturally encoded amino acid is incorporated atposition 7 in the B chain (SEQ ID NO: 5 or SEQ ID NO: 6, or thecorresponding amino acid positions in SEQ ID NOs: 1, 2, or 3). In someembodiments, a non-naturally encoded amino acid is incorporated atposition 5 in the B chain (SEQ ID NO: 5 or SEQ ID NO: 6, or thecorresponding amino acid positions in SEQ ID NOs: 1, 2, or 3). In someembodiments, a non-naturally encoded amino acid is incorporated atposition 7 in the B chain (SEQ ID NO: 5 or SEQ ID NO: 6, or thecorresponding amino acid positions in SEQ ID NOs: 1, 2, or 3).

In one embodiments, a non-naturally encoded amino acids is incorporatedin one of the following positions in the relaxin polypeptides: in the Achain at amino acid positions 1, 5, 2, 13, 18 (SEQ ID NO: 4 orcorresponding amino acid positions in SEQ ID NO. 1, 2, 3), in the Bchain at amino acid positions 7, 5 (SEQ ID NO: 5 or 6, or correspondingamino acid positions in SEQ ID NO: 1, 2.3). In some embodiments, one ormore non-naturally encoded amino acids are incorporated in one of thefollowing positions in the relaxin polypeptides: in the A chain at aminoacid positions 1, 5, 2, 13, 18 (SEQ ID NO: 4 or corresponding amino acidpositions in SEQ ID NO. 1, 2, 3), in the B chain at amino acid positions7, 5 (SEQ ID NO: 5 or 6, or corresponding amino acid positions in SEQ IDNO: 1, 2, 3).

In some embodiments, the non-naturally encoded amino acid at one or moreof these positions is linked to a water soluble polymer, including butnot limited to, positions: in the A chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 (i.e., at the carboxyl terminus of theprotein), and any combination thereof (SEQ ID NO: 4 or the correspondingamino acids in known relaxin sequences) and/or in the B chain beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 (SEQ ID NO: 5 or 6 or the corresponding amino acids in known relaxinsequences). In some embodiments, the non-naturally encoded amino acid atone or more of these positions is linked to a water soluble polymer,including but not limited to: in the A chain 1, 2, 5, 13, 18 (SEQ ID NO:1 or the corresponding amino acids in known relaxin sequences). In someembodiments, the non-naturally encoded amino acid at one or more ofthese positions is linked to a water soluble polymer, including but notlimited to: in the A chain 1, 2, 5 (SEQ ID NO: 1 or the correspondingamino acids in known relaxin sequences). In some embodiments, thenon-naturally encoded amino acid at one or more of these positions islinked to a water soluble polymer, including but not limited to: in theA chain 2, 5 (SEQ ID NO: 1 or the corresponding amino acids in knownrelaxin sequences). In some embodiments, the non-naturally encoded aminoacid at one or more of these positions is linked to a water solublepolymer, including but not limited to: in the A chain 2, 5, 13, 18 (SEQID NO: 1 or the corresponding amino acids in known relaxin sequences).In some embodiments, the non-naturally encoded amino acid at one or moreof these positions is linked to a water soluble polymer, including butnot limited to: in the B chain 5, 7 (SEQ ID NO: 5 or 6 or thecorresponding amino acid positions in SEQ ID NOs: 1, 2, 3). In someembodiments, the non-naturally encoded amino acid at position 5 in the Bchain (SEQ ID NO: 5 or 6 or the corresponding amino acid positions inSEQ ID NOs: 1, 2, 3) is linked to a water soluble polymer. In someembodiments, the non-naturally encoded amino acid at position 7 in the Bchain (SEQ ID NO: 5 or 6 or the corresponding amino acid positions inSEQ ID NOs: 1, 2, 3) is linked to a water soluble polymer

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of therelaxin or prorelaxin polypeptides: B chain positions 5, 7 (SEQ ID NO: 5or 6 or the corresponding amino acid positions in SEQ ID NOs: 1, 2, 3)and A chain positions 1, 5, 2, 13, 18 (SEQ ID NO: 4 or the correspondingamino acid positions in SEQ ID NOs: 1, 2, 3). In some embodiments, oneor more non-naturally encoded amino acids are incorporated in one ormore of the following positions in any of the relaxin or prorelaxinpolypeptides: B chain positions 5, 7 (SEQ ID NO: 5 or 6 or thecorresponding amino acid positions in SEQ ID NOs: 1, 2, 3) and A chainpositions 1, 5, 2, 13, 18 (SEQ ID NO: 4 or the corresponding amino acidpositions in SEQ ID NOs: 1, 2, 3) and the non-naturally encoded aminoacid is linked to a water soluble polymer.

Methods of the present invention could be used to promote angiogenesis,promote vasodilation, promote non-hypotensive vasodilation, to treathypertension, including but not limited to renal hypertension, pulmonaryhypertension, and cardiac hypertension (U.S. Pat. Nos. 6,723,702; and6,780,836 both hereby incorporated by reference in their entirety),discloses formation and use of crystals of a relaxin analog.

Formulations

In the broad practice of the present invention, it also is contemplatedthat a formulation may contain a mixture of two or more of a relaxin, arelaxin dimer, a relaxin analog, an acylated relaxin, or acylatedrelaxin analog with at least one of the components of the mixturecontaining a non-naturally encoded amino acid. In another embodiment ofthe present invention, the formulations containing a mixture of two ormore of relaxin, a relaxin analog, an acylated relaxin, or acylatedrelaxin analog with at least one of the components of the mixturecontaining a non-naturally encoded amino acid also includes at least onewater soluble polymer attached to at least one of the non-naturallyencoded amino acids.

The present invention also includes heterogeneous mixtures whereinrelaxin polypeptides and relaxin analogs are prepared by the methodsdisclosed in this invention and are then mixed so that a formulation maybe administered to a patient in need thereof which contains, forexample, 25% relaxin polypeptide containing a non-naturally encodedamino acid at position 28 of the B chain which has been pegylated, 25%relaxin polypeptide containing a non-naturally encoded amino acid atposition 10 of the B chain, said non-naturally encoded amino acidcoupled to a water soluble polymer, and 50% relaxin polypeptide whereina non-naturally encoded amino acid occurs at position 31 of the B chainof relaxin (SEQ ID NO: 2; alternatively SEQ ID NOs: 4, 6, 8, 10, or 12).All different mixtures of different percentage amounts of relaxinpolypeptide variants wherein the relaxin polypeptides include a variety(1) with differently sized PEGs, or (2) PEGs are included at differentpositions in the sequence. This is intended as an example and should inno way be construed as limiting to the formulations made possible by thepresent invention and will be apparent to those of skill in the art. Inan additional embodiment, the relaxin polypeptide variants to include inthe formulation mixture will be chosen by their varying dissociationtimes so that the formulation may provide a sustained release of relaxinfor a patient in need thereof.

Formulations of the present invention may include a glucagon.

Other embodiments of the present invention including formulation forinhalation

In an additional embodiment of the present invention, it is possible touse the technology disclosed herein for the production of relaxinanalogs with increased pharmacokinetic and pharmacodynamic propertiesfor patient use via administration to the lung, resulting in elevatedblood levels of relaxin that are sustained for at least 6 hours, andmore typically for at least 8, 10, 12, 14, 18, 24 hours or greaterpost-administration. Another embodiment of the present invention allowsfor advantageous mixtures of relaxin analogs suitable for therapeuticformulations designed to be administered to patients as an inhalant.

In some embodiments of the present invention, the following sites in thenative relaxin molecule may be substituted with non-naturally encodedamino acids and optionally further modified by covalent attachment of awater soluble polymer, such as PEG: the 2 C-termini of the A and Bchains, positions 1, 5, 18, 13, 2 of SEQ ID NO: 4 or in those positionsof the A chain of any of the known relaxin sequences or 5, 7, 18, 28 ofSEQ ID NO: 5 or 6 in those same positions of the B chain of any of theknown relaxin sequences.

In addition to native relaxin, the present invention provides fornon-native relaxin polypeptides and relaxin analogs having one or morenon-naturally encoded amino acids substituted or inserted into thesignal sequence that may also provide a site for the incorporation ofone or more water soluble polymers, such as PEG. This embodiment of theinvention is particularly useful for introducing additional, customizedpegylation-sites within the relaxin molecule, for example, for forming aPEG-relaxin having improved resistance to enzymatic degradation. Such anapproach provides greater flexibility in the design of an optimizedrelaxin conjugate having the desired balance of activity, stability,solubility, and pharmacological properties. Mutations can be carriedout, i.e., by site specific mutagenesis, at any number of positionswithin the relaxin molecule. PEGs for use in the present invention maypossess a variety of structures: linear, forked, branched, dumbbell, andthe like. Typically, PEG is activated with a suitable activating groupappropriate for coupling a desired site or sites on the relaxinmolecule. An activated PEG will possess a reactive group at a terminusfor reaction with relaxin. Representative activated PEG derivatives andmethods for conjugating these agents to a drug such as relaxin are knownin the art and further described in Zalipsky, S., et al., “Use ofFunctionalized Poly(Ethylene Glycols) for Modification of Polypeptides”in Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, Plenus Press, New York (1992), and inAdvanced Drug Reviews, 16:157-182 (1995).

In one particular embodiment of the invention, the PEG portion of theconjugate is absent one or more lipophilic groups effective tosignificantly modify the water-soluble nature of the polymer or of thepolymer-relaxin conjugate. That is to say, the polymer or non-relaxinportion of a conjugate of the invention may contain a group of atomsconsidered to be more lipophilic than hydrophilic (e.g., a carbon chainhaving from about 2 to 8-12 carbon atoms), however, if the presence ofsuch a group or groups is not effective to significantly alter thehydrophilic nature of the polymer or of the conjugate, then such amoiety may be contained in the conjugates of the invention. That is tosay, through site-specific mutations of relaxin, relaxin polypeptides,and relaxin analogs, a relaxin conjugate of the invention itself mayexhibit hydrophilic, rather than lipophilic or amphiphilic. In certainembodiments of the invention where a lipophilic moiety may be present,the moiety is preferably not positioned at a terminus of a PEG chain.

Branched PEGs for use in the conjugates of the invention include thosedescribed in International Patent Publication WO 96/21469, the contentsof which is expressly incorporated herein by reference in its entirety.Generally, branched PEGs can be represented by the formulaR(PEG-OH)_(n), where R represents the central “core” molecule and nrepresents the number of arms. Branched PEGs have a central core fromwhich extend 2 or more “PEG” arms. In a branched configuration, thebranched polymer core possesses a single reactive site for attachment torelaxin. Branched PEGs for use in the present invention will typicallycomprise fewer than 4 PEG arms, and more preferably, will comprise fewerthan 3 PEG arms. Branched PEGs offer the advantage of having a singlereactive site, coupled with a larger, more dense polymer cloud thantheir linear PEG counterparts. One particular type of branched PEG canbe represented as (MeO-PEG-)_(p) R—X, where p equals 2 or 3, R is acentral core structure such as lysine or glycerol having 2 or 3 PEG armsattached thereto, and X represents any suitable functional group that isor that can be activated for coupling to relaxin. One particularlypreferred branched PEG is mPEG2-NHS (Shearwater Corporation, Alabama)having the structure mPEG2-lysine-succinimide.

In yet another branched architecture, “pendant PEG” has reactive groupsfor protein coupling positioned along the PEG backbone rather than atthe end of PEG chains. The reactive groups extending from the PEGbackbone for coupling to relaxin may be the same or different. PendantPEG structures may be useful but are generally less preferred,particularly for compositions for inhalation.

Alternatively, the PEG-portion of a PEG-relaxin conjugate may possess aforked structure having a branched moiety at one end of the polymerchain and two free reactive groups (or any multiple of 2) linked to thebranched moiety for attachment to relaxin. Exemplary forked PEGs aredescribed in International Patent Publication No. WO 99/45964, thecontent of which is expressly incorporated herein by reference. Theforked polyethylene glycol may optionally include an alkyl or “R” groupat the opposing end of the polymer chain. More specifically, a forkedPEG-relaxin conjugate in accordance with the invention has the formula:R-PEG-L(Y-relaxin)n where R is alkyl, L is a hydrolytically stablebranch point and Y is a linking group that provides chemical linkage ofthe forked polymer to relaxin, and n is a multiple of 2. L may representa single “core” group, such as “—CH—”, or may comprise a longer chain ofatoms. Exemplary L groups include lysine, glycerol, pentaerythritol, orsorbitol. Typically, the particular branch atom within the branchingmoiety is carbon.

In one particular embodiment of the invention, the linkage of the forkedPEG to the relaxin molecule, (Y), is hydrolytically stable. In apreferred embodiment, n is 2. Suitable Y moieties, prior to conjugationwith a reactive site on relaxin, include but are not limited to activeesters, active carbonates, aldehydes, isocyanates, isothiocyanates,epoxides, alcohols, maleimides, vinylsulfones, hydrazides,dithiopyridines, and iodacetamides. Selection of a suitable activatinggroup will depend upon the intended site of attachment on the relaxinmolecule and can be readily determined by one of skill in the art. Thecorresponding Y group in the resulting PEG-relaxin conjugate is thatwhich results from reaction of the activated forked polymer with asuitable reactive site on relaxin. The specific identity of such thefinal linkage will be apparent to one skilled in the art. For example,if the reactive forked PEG contains an activated ester, such as asuccinimide or maleimide ester, conjugation via an amine site on relaxinwill result in formation of the corresponding amide linkage. Theseparticular forked polymers are particularly attractive since theyprovide conjugates having a molar ratio of relaxin to PEG of 2:1 orgreater. Such conjugates may be less likely to block the relaxinreceptor site, while still providing the flexibility in design toprotect the relaxin against enzymatic degradation, e.g., by relaxindegrading enzyme.

In a related embodiment, a forked PEG-relaxin conjugate may be used inthe present invention, represented by the formula:R-[PEG-L(Y-relaxin)2]n. In this instance R represents a non-naturallyencoded amino acid having attached thereto at least one PEG-di-relaxinconjugate. Specifically, preferred forked polymers in accordance withthis aspect of the invention are those were n is selected from the groupconsisting of 1, 2, 3, 4, 5, and 6. In an alternative embodiment, thechemical linkage between the non-natural amino acid within relaxin,relaxin polypeptide, or relaxin analog and the polymer branch point maybe degradable (i.e., hydrolytically unstable). Alternatively, one ormore degradable linkages may be contained in the polymer backbone toallow generation in vivo of a PEG-relaxin conjugate having a smaller PEGchain than in the initially administered conjugate. For example, a largeand relatively inert conjugate (i.e., having one or more high molecularweight PEG chains attached thereto, e.g., one or more PEG chains havinga molecular weight greater than about 10,000, wherein the conjugatepossesses essentially no bioactivity) may be administered, which theneither in the lung or in the bloodstream, is hydrolyzed to generate abioactive conjugate possessing a portion of the originally present PEGchain. Upon in-vivo cleavage of the hydrolytically degradable linkage,either free relaxin (depending upon the position of the degradablelinkage) or relaxin having a small polyethylene tag attached thereto, isthen released and more readily absorbed through the lung and/orcirculated in the blood.

In one feature of this embodiment of the invention, the intactpolymer-conjugate, prior to hydrolysis, is minimally degraded uponadministration, such that hydrolysis of the cleavable bond is effectiveto govern the slow rate of release of active relaxin into thebloodstream, as opposed to enzymatic degradation of relaxin prior to itsrelease into the systemic circulation.

Appropriate physiologically cleavable linkages include but are notlimited to ester, carbonate ester, carbamate, sulfate, phosphate,acyloxyalkyl ether, acetal, and ketal. Such conjugates should possess aphysiologically cleavable bond that is stable upon storage and uponadministration. For instance, a PEG-cleavable linkage-relaxin conjugateshould maintain its integrity upon manufacturing of the finalpharmaceutical composition, upon dissolution in an appropriate deliveryvehicle, if employed, and upon administration irrespective of route.

Thus, in another embodiment of the present invention, one or morenon-naturally encoded amino acids are incorporated into a single chainrelaxin or single chain relaxin analog.

In some embodiments, the polypeptide of the invention comprises one ormore non-naturally encoded amino acid substitution, addition, ordeletion in the signal sequence. In some embodiments, the polypeptide ofthe invention comprises one or more non-naturally encoded amino acidsubstitution, addition, or deletion in the signal sequence for relaxinor any of the relaxin analogs or polypeptides disclosed within thisspecification. In some embodiments, the polypeptide of the inventioncomprises one or more naturally encoded amino acid substitution,addition, or deletion in the signal sequence as well as one or morenon-naturally encoded amino acid substitutions, additions, or deletionsin the signal sequence for relaxin or any of the relaxin analogs orpolypeptides disclosed within this specification. In some embodiments,one or more non-natural amino acids are incorporated in the leader orsignal sequence for relaxin or any of the relaxin analogs orpolypeptides disclosed within this specification.

In some embodiments, the relaxin polypeptide comprises a substitution,addition or deletion that modulates affinity of the relaxin polypeptidefor a relaxin polypeptide receptor or binding partner, including but notlimited to, a protein, polypeptide, small molecule, or nucleic acid. Insome embodiments, the relaxin polypeptide comprises a substitution,addition, or deletion that increases the stability of the relaxinpolypeptide when compared with the stability of the correspondingrelaxin without the substitution, addition, or deletion. Stabilityand/or solubility may be measured using a number of different assaysknown to those of ordinary skill in the art. Such assays include but arenot limited to SE-HPLC and RP-HPLC. In some embodiments, the relaxinpolypeptide comprises a substitution, addition, or deletion thatmodulates the immunogenicity of the relaxin polypeptide when comparedwith the immunogenicity of the corresponding relaxin without thesubstitution, addition, or deletion. In some embodiments, the relaxinpolypeptide comprises a substitution, addition, or deletion thatmodulates serum half-life or circulation time of the relaxin polypeptidewhen compared with the serum half-life or circulation time of thecorresponding relaxin without the substitution, addition, or deletion.

In some embodiments, the relaxin polypeptide comprises a substitution,addition, or deletion that increases the aqueous solubility of therelaxin polypeptide when compared to aqueous solubility of thecorresponding relaxin without the substitution, addition, or deletion.In some embodiments, the relaxin polypeptide comprises a substitution,addition, or deletion that increases the solubility of the relaxinpolypeptide produced in a host cell when compared to the solubility ofthe corresponding relaxin without the substitution, addition, ordeletion. In some embodiments, the relaxin polypeptide comprises asubstitution, addition, or deletion that increases the expression of therelaxin polypeptide in a host cell or increases synthesis in vitro whencompared to the expression or synthesis of the corresponding relaxinwithout the substitution, addition, or deletion. The relaxin polypeptidecomprising this substitution retains agonist activity and retains orimproves expression levels in a host cell. In some embodiments, therelaxin polypeptide comprises a substitution, addition, or deletion thatincreases protease resistance of the relaxin polypeptide when comparedto the protease resistance of the corresponding relaxin without thesubstitution, addition, or deletion. In some embodiments, the relaxinpolypeptide comprises a substitution, addition, or deletion thatmodulates signal transduction activity of the relaxin receptor whencompared with the activity of the receptor upon interaction with thecorresponding relaxin polypeptide without the substitution, addition, ordeletion. In some embodiments, the relaxin polypeptide comprises asubstitution, addition, or deletion that modulates its binding toanother molecule such as a receptor when compared to the binding of thecorresponding relaxin polypeptide without the substitution, addition, ordeletion. In some embodiments, the relaxin polypeptide comprises asubstitution, addition, or deletion that modulates its anti-viralactivity compared to the anti-viral activity of the correspondingrelaxin polypeptide without the substitution, addition, or deletion.

In some embodiments, the relaxin polypeptide comprises a substitution,addition, or deletion that increases compatibility of the relaxinpolypeptide with pharmaceutical preservatives (e.g., m-cresol, phenol,benzyl alcohol) when compared to compatibility of the correspondingrelaxin without the substitution, addition, or deletion. This increasedcompatibility would enable the preparation of a preserved pharmaceuticalformulation that maintains the physiochemical properties and biologicalactivity of the protein during storage.

In some embodiments, one or more engineered bonds are created with oneor more non-natural amino acids. The intramolecular bond may be createdin many ways, including but not limited to, a reaction between two aminoacids in the protein under suitable conditions (one or both amino acidsmay be a non-natural amino acid); a reaction with two amino acids, eachof which may be naturally encoded or non-naturally encoded, with alinker, polymer, or other molecule under suitable conditions; etc.

In some embodiments, one or more amino acid substitutions in the relaxinpolypeptide may be with one or more naturally occurring or non-naturallyencoded amino acids. In some embodiments the amino acid substitutions inthe relaxin polypeptide may be with naturally occurring or non-naturallyencoded amino acids, provided that at least one substitution is with anon-naturally encoded amino acid. In some embodiments, one or more aminoacid substitutions in the relaxin polypeptide may be with one or morenaturally occurring amino acids, and additionally at least onesubstitution is with a non-naturally encoded amino acid.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an acetyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10: R1 is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R2 is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R3 is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R4 is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R2 is H, anamino acid, a polypeptide, or an amino terminus modification group, andR3 is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R2 is H, anamino acid, a polypeptide, or an amino terminus modification group, andR3 is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the polypeptide is a relaxin polypeptide agonist,partial agonist, antagonist, partial antagonist, or inverse agonist. Insome embodiments, the relaxin polypeptide agonist, partial agonist,antagonist, partial antagonist, or inverse agonist comprises anon-naturally encoded amino acid linked to a water soluble polymer. Insome embodiments, the water soluble polymer comprises a poly(ethyleneglycol) moiety. In some embodiments, the relaxin polypeptide agonist,partial agonist, antagonist, partial antagonist, or inverse agonistcomprises a non-naturally encoded amino acid and one or morepost-translational modification, linker, polymer, or biologically activemolecule.

The present invention also provides isolated nucleic acids comprising apolynucleotide that hybridizes under stringent conditions nucleic acidsthat encode relaxin polypeptides of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, and 12. The present invention also provides isolated nucleicacids comprising a polynucleotide that hybridizes under stringentconditions nucleic acids that encode relaxin polypeptides of SEQ ID NOs:1 and 2. The present invention also provides isolated nucleic acidscomprising a polynucleotide or polynucleotides that hybridize understringent conditions to polynucleotides that encode polypeptides shownas SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 wherein thepolynucleotide comprises at least one selector codon. The presentinvention also provides isolated nucleic acids comprising apolynucleotide or polynucleotides that hybridize under stringentconditions to polynucleotides that encode polypeptides shown as SEQ IDNOs: 1 and 2 wherein the polynucleotide comprises at least one selectorcodon. The present invention provides isolated nucleic acids comprisinga polynucleotide that encodes the polypeptides shown as SEQ ID NOs.: 1and 2. The present invention also provides isolated nucleic acidscomprising a polynucleotide that encodes the polypeptides shown as SEQID NOs.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The presentinvention provides isolated nucleic acids comprising a polynucleotidethat encodes the polypeptides shown as SEQ ID NOs.: 1 and 2 with one ormore non-naturally encoded amino acids. The present invention alsoprovides isolated nucleic acids comprising a polynucleotide that encodesthe polypeptides shown as SEQ ID NOs.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, and 12 with one or more non-naturally encoded amino acids. It isreadily apparent to those of ordinary skill in the art that a number ofdifferent polynucleotides can encode any polypeptide of the presentinvention.

In some embodiments, the selector codon is selected from the groupconsisting of an amber codon, ochre codon, opal codon, a unique codon, arare codon, a five-base codon, and a four-base codon.

The present invention also provides methods of making a relaxinpolypeptide linked to a water soluble polymer. In some embodiments, themethod comprises contacting an isolated relaxin polypeptide comprising anon-naturally encoded amino acid with a water soluble polymer comprisinga moiety that reacts with the non-naturally encoded amino acid. In someembodiments, the non-naturally encoded amino acid incorporated into therelaxin polypeptide is reactive toward a water soluble polymer that isotherwise unreactive toward any of the 20 common amino acids. In someembodiments, the non-naturally encoded amino acid incorporated into therelaxin polypeptide is reactive toward a linker, polymer, orbiologically active molecule that is otherwise unreactive toward any ofthe 20 common amino acids.

In some embodiments, the relaxin polypeptide linked to the water solublepolymer is made by reacting a relaxin polypeptide comprising acarbonyl-containing amino acid with a poly(ethylene glycol) moleculecomprising an aminooxy, hydrazine, hydrazide or semicarbazide group. Insome embodiments, the aminooxy, hydrazine, hydrazide or semicarbazidegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage. In some embodiments, the aminooxy, hydrazine, hydrazide orsemicarbazide group is linked to the poly(ethylene glycol) moleculethrough a carbamate linkage.

In some embodiments, the relaxin polypeptide linked to the water solublepolymer is made by reacting a poly(ethylene glycol) molecule comprisinga carbonyl group with a polypeptide comprising a non-naturally encodedamino acid that comprises an aminooxy, hydrazine, hydrazide orsemicarbazide group.

In some embodiments, the relaxin polypeptide linked to the water solublepolymer is made by reacting a relaxin polypeptide comprising analkyne-containing amino acid with a poly(ethylene glycol) moleculecomprising an azide moiety. In some embodiments, the azide or alkynegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the relaxin polypeptide linked to the water solublepolymer is made by reacting a relaxin polypeptide comprising anazide-containing amino acid with a poly(ethylene glycol) moleculecomprising an alkyne moiety. In some embodiments, the azide or alkynegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the poly(ethylene glycol) molecule has a molecularweight of between about 0.1 kDa and about 100 kDa. In some embodiments,the poly(ethylene glycol) molecule has a molecular weight of between 0.1kDa and 50 kDa.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. In some embodiments, each branch of the poly(ethylene glycol)branched polymer has a molecular weight of between 1 kDa and 100 kDa, orbetween 1 kDa and 50 kDa.

In some embodiments, the water soluble polymer linked to the relaxinpolypeptide comprises a polyalkylene glycol moiety. In some embodiments,the non-naturally encoded amino acid residue incorporated into therelaxin polypeptide comprises a carbonyl group, an aminooxy group, ahydrazide group, a hydrazine, a semicarbazide group, an azide group, oran alkyne group. In some embodiments, the non-naturally encoded aminoacid residue incorporated into the relaxin polypeptide comprises acarbonyl moiety and the water soluble polymer comprises an aminooxy,hydrazide, hydrazine, or semicarbazide moiety. In some embodiments, thenon-naturally encoded amino acid residue incorporated into the relaxinpolypeptide comprises an alkyne moiety and the water soluble polymercomprises an azide moiety. In some embodiments, the non-naturallyencoded amino acid residue incorporated into the relaxin polypeptidecomprises an azide moiety and the water soluble polymer comprises analkyne moiety.

The present invention also provides compositions comprising a relaxinpolypeptide comprising a non-naturally encoded amino acid and apharmaceutically acceptable carrier. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.

The present invention also provides cells comprising a polynucleotideencoding the relaxin polypeptide comprising a selector codon. In someembodiments, the cells comprise an orthogonal RNA synthetase and/or anorthogonal tRNA for substituting a non-naturally encoded amino acid intothe relaxin polypeptide.

The present invention also provides methods of making a relaxinpolypeptide comprising a non-naturally encoded amino acid. In someembodiments, the methods comprise culturing cells comprising apolynucleotide or polynucleotides encoding a relaxin polypeptide, anorthogonal RNA synthetase and/or an orthogonal tRNA under conditions topermit expression of the relaxin polypeptide; and purifying the relaxinpolypeptide from the cells and/or culture medium.

The present invention also provides methods of increasing therapeutichalf-life, serum half-life or circulation time of relaxin polypeptides.The present invention also provides methods of modulating immunogenicityof relaxin polypeptides. In some embodiments, the methods comprisesubstituting a non-naturally encoded amino acid for any one or moreamino acids in naturally occurring relaxin polypeptides and/or linkingthe relaxin polypeptide to a linker, a polymer, a water soluble polymer,or a biologically active molecule.

The present invention also provides methods of treating a patient inneed of such treatment with an effective amount of a relaxin molecule ofthe present invention. In some embodiments, the methods compriseadministering to the patient a therapeutically-effective amount of apharmaceutical composition comprising a relaxin polypeptide comprising anon-naturally-encoded amino acid and a pharmaceutically acceptablecarrier. In some embodiments, the non-naturally encoded amino acid islinked to a water soluble polymer. In some embodiments, the relaxinpolypeptide is glycosylated. In some embodiments, the relaxinpolypeptide is not glycosylated.

The present invention also provides relaxin polypeptides comprising asequence shown in SEQ ID NO: 1 and 2, or any other relaxin polypeptidesequence (a non-limiting example of these would be SEQ ID NOs: 3 through12) except that at least one amino acid is substituted by anon-naturally encoded amino acid. In some embodiments, the non-naturallyencoded amino acid is linked to a water soluble polymer. In someembodiments, the water soluble polymer comprises a poly(ethylene glycol)moiety. In some embodiments, the non-naturally encoded amino acidcomprises a carbonyl group, an aminooxy group, a hydrazide group, ahydrazine group, a semicarbazide group, an azide group, or an alkynegroup.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a relaxinpolypeptide comprising the sequence shown in SEQ ID NOs: 1 through 12,or any other relaxin polypeptide sequence, wherein at least one aminoacid is substituted by a non-naturally encoded amino acid. The presentinvention also provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and a relaxin polypeptide comprisingan A and B chain (e.g. SEQ ID NO: 1, 2, and 3; SEQ ID NOs: 4 and 5 or 4and 6 would make relaxin, etc.), or any other relaxin polypeptidesequence, wherein at least one amino acid is substituted by anon-naturally encoded amino acid. The present invention also providespharmaceutical compositions comprising a pharmaceutically acceptablecarrier and a relaxin polypeptide comprising the sequence shown in SEQID NO: 1, 2, and/or 3. The present invention also providespharmaceutical compositions comprising a pharmaceutically acceptablecarrier and a relaxin polypeptide comprising the sequence shown in SEQID NO: 1-3. In some embodiments, the non-naturally encoded amino acidcomprises a saccharide moiety. In some embodiments, the water solublepolymer is linked to the polypeptide via a saccharide moiety. In someembodiments, a linker, polymer, or biologically active molecule islinked to the relaxin polypeptide via a saccharide moiety.

The present invention also provides a relaxin polypeptide comprising awater soluble polymer linked by a covalent bond to the relaxinpolypeptide at a single amino acid. In some embodiments, the watersoluble polymer comprises a poly(ethylene glycol) moiety. In someembodiments, the amino acid covalently linked to the water solublepolymer is a non-naturally encoded amino acid present in thepolypeptide.

The present invention provides a relaxin polypeptide comprising at leastone linker, polymer, or biologically active molecule, wherein saidlinker, polymer, or biologically active molecule is attached to thepolypeptide through a functional group of a non-naturally encoded aminoacid ribosomally incorporated into the polypeptide. In some embodiments,the polypeptide is monoPEGylated. The present invention also provides arelaxin polypeptide comprising a linker, polymer, or biologically activemolecule that is attached to one or more non-naturally encoded aminoacid wherein said non-naturally encoded amino acid is ribosomallyincorporated into the polypeptide at pre-selected sites.

Included within the scope of this invention is the relaxin leader orsignal sequence an example of which can be seen as proinulin. Theheterologous leader or signal sequence selected should be one that isrecognized and processed, e.g. by host cell secretion system to secreteand possibly cleaved by a signal peptidase, by the host cell. A methodof treating a condition or disorder with relaxin or a relaxinpolypeptide or analog of the present invention is meant to implytreating with relaxin with or without a signal or leader peptide.

In another embodiment, conjugation of the relaxin polypeptide comprisingone or more non-naturally encoded amino acids to another molecule,including but not limited to PEG, provides substantially purifiedrelaxin due to the unique chemical reaction utilized for conjugation tothe non-natural amino acid. Conjugation of relaxin comprising one ormore non-naturally encoded amino acids to another molecule, such as PEG,may be performed with other purification techniques performed prior toor following the conjugation step to provide substantially pure relaxin.

In a further aspect, the invention provides recombinant nucleic acidsencoding the variant proteins, expression vectors containing the variantnucleic acids, host cells comprising the variant nucleic acids and/orexpression vectors, and methods for producing the variant proteins. Inan additional aspect, the invention provides treating a relaxinresponsive disorder by administering to a patient a variant protein,usually with a pharmaceutical carrier, in a therapeutically effectiveamount. In a further aspect, the invention provides methods formodulating immunogenicity (particularly reducing immunogenicity) ofrelaxin polypeptides by altering MHC Class II epitopes.

In therapeutic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

In prophylactic applications, compositions containing the relaxinpolypeptide are administered to a patient susceptible to or otherwise atrisk of a particular disease, disorder or condition. Such an amount isdefined to be a “prophylactically effective amount.” In this use, theprecise amounts also depend on the patient's state of health, weight,and the like. It is considered well within the skill of the art for oneto determine such prophylactically effective amounts by routineexperimentation (e.g., a dose escalation clinical trial).

Relaxin polypeptides of the present invention can be used to modulatevasoconstriction, NO production, ET-1, Ang II, and platelet aggregation.In one embodiment of the present invention, a patient in need thereofreceives a therapeutic amount of relaxin polypeptides of the presentinvention that would decrease the patient's vasoconstriction over thebaseline of their seeking treatment by 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50% 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, morethan 100%, 150%, more than 150%, 200%, more than 200%. In anotherembodiment of the present invention is a method of treatment of apatient in need thereof to increase the patient's NO production byadministering a therapeutically effective amount of relaxin polypeptideto increase NO production by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 450%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, more than 100%,150%, more than 150%, 200%, more than 200%.

In one embodiment of the present invention is a method of treatment of apatient in need thereof with a therapeutic amount of relaxinpolypeptides of the present invention that decreases the patient'splatelet aggregation by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, more than 100%, 150%,more than 150%, 200%, more than 200%. In another embodiment of thepresent invention is a method of treatment of a patient in need thereofwith a therapeutic amount of relaxin polypeptides to decreasehypertrophy. In another embodiment of the present invention is a methodof treatment of a patient in need thereof with a therapeutic amount ofrelaxin polypeptides of the present invention that decreases thepatient's CF-stimulated protein synthesis by 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,more than 100%, 15%, more than 150%, 200%, more than 200%. In anotherembodiment of the present invention is a method of treatment of apatient in need thereof to increase the patient's ANP expression byadministering a therapeutically effective amount of relaxin polypeptideto increase NO production by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, more than 100%,150%, more than 150%, 200%, more than 200%.

Present methods whereby a peg-relaxin of the present invention has a10-fold increase in AUC as compared to a wild type relaxin 15-foldincrease; more than 15-fold increase; 20-fold increase; more than20-fold increase; 25 fold increase; more than 25-fold increase; 30-foldincrease; more than 30-fold increase; 35-fold increase; more than35-fold increase: 40-fold increase; more than 40-fold increase; 45-foldincrease; more than 45-fold increase; 50-fold increase; more than50-fold increase; 55-fold increase; more than 55-fold increase; 60-foldincrease; more than 60-fold increase; 65-fold increase; more than65-fold increase; 70-fold increase; more than 70-fold increase; 75-foldincrease; more than 75-fold increase; 80-fold increase; more than80-fold increase; 85-fold increase; more than 85-fold increase; 90-foldincrease; more than 90-fold increase; 95-fold increase; more than95-fold increase; 100-fold increase; more than 100-fold increase.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a model of the crystal structure of relaxin are shown alongwith some amino acid residue positions selected for substitution.

FIG. 2 is a model of the crystal structure of relaxin are shown alongwith some amino acid residue positions selected for substitution.

FIG. 3 is a model of the crystal structure of relaxin are shown alongwith some amino acid residue positions selected for substitution.

FIG. 4 is a drawing of the structure of the A and B chain of humanrelaxin.

FIG. 5 shows an SDS-PAGE gel of the prorelaxin produced by these methodswith a chain B1 amino acid as Ala and a para-acetyl phenylalanine in the13^(th) amino acid position of the A chain, substituted for valine.

FIG. 6A shows an SDS-PAGE gel of unPEGylated relaxin V13pAF alongside amolecular weight marker in lane 1 and recombinant and non-recombinant WTrelaxin in lanes 3 and 7 (non-reduced (NR) and reduced (R)). FIG. 6Bshows an SDS-PAGE gel of PEGylated relaxin V13pAF in lanes 3 and 4(non-reduced (NR) and reduced (R)) alongside a molecular weight markerin lane 1.

FIG. 7 shows a graph of SD rat serum relaxin concentration in ng/mL overtime for differently PEGylated AV13 and wild type relaxin polypeptides.

FIG. 8A shows a graph of the comparison of group mean serumconcentration versus time for all PEG-RLX groups dosed subcutaneously inExample 40. A single dose injection was administered to each animal. N=5animals per group. Symbols indicate mean±SD of grouped serumconcentrations versus time.

FIG. 8B shows a graph of the individual animal serum concentration timecurves for SD rats dosed subcutaneously with 0.5 mg/kg of20KPEG-AQ1-RLX. A single, subcutaneous dose was administered to eachanimal. N=5 animals per group.

FIG. 9A shows a graph of the individual animal serum concentration timecurves for SD rats dosed subcutaneously with 0.5 mg/kg of PEG20-AA5-RLX.A single, subcutaneous dose was administered to each animal. N=5 animalsper group.

FIG. 9B shows a graph of the individual animal serum concentration timecurves for SD rats dosed subcutaneously with 0.5 mg/kg ofPEG20-AR18-RLX. A single, subcutaneous dose was administered to eachanimal. N=5 animals per group.

FIG. 10A shows a graph of the individual animal serum concentration timecurves for SD rats dosed intravenously with 0.5 mg/kg of PEG20-BV7-RLX.A single, intravenous dose was administered to each animal. N=5 animalsper group.

FIG. 10B shows a graph of the individual animal serum concentration timecurves for SD rats dosed intravenously with 0.5 mg/kg of PEG20-BW28-RLX.A single, intravenous dose was administered to each animal. N=5 animalsper group.

FIG. 11 shows a graph of the individual animal serum concentration timecurves for SD rats dosed intravenously with 0.5 mg/kg of PEG20-AV13-RLX.A single, intravenous dose was administered to each animal. N=5 animalsper group.

FIG. 12A shows a graph of a comparison of group mean serum concentrationversus time for wt rhRelaxin dosed subcutaneously. A single doseinjection was administered to each animal. N=5 animals per group.

FIG. 12B shows a graph of individual animal serum concentration timecurves for SD rats dosed intravenously with 0.5 mg/kg of wt rhRelaxin. Asingle, intravenous dose was administered to each animal. N=5 animalsper group.

FIG. 13A shows a comparison of group mean serum concentration versustime for all PEG-RLX groups dosed subcutaneously or intravenously. Asingle dose injection was administered to each animal. N=3-5 animals pergroup. MPK: milligrams per kilogram.

FIG. 13B shows a graph of individual animal serum concentration timecurves for SD rats dosed intravenously with 0.25 mg/kg of20KPEG-AQ1-RLX. A single, intravenous dose was administered to eachanimal. N=4 animals per group. MPK: milligrams per kilogram.

FIG. 14A shows a graph of individual animal serum concentration timecurves for SD rats dosed subcutaneously with 0.5 mg/kg of PEG20-AQ1-RLX.A single, subcutaneous dose was administered to each animal. N=5 animalsper group. MPK: milligrams per kilogram.

FIG. 14B shows a graph of individual animal serum concentration timecurves for SD rats dosed subcutaneously with 0.25 mg/kg ofPEG20-AQ1-RLX. A single, subcutaneous dose was administered to eachanimal. N=3 animals per group. MPK: milligrams per kilogram.

FIG. 15 shows a graph of individual animal serum concentration timecurves for SD rats dosed intravenously with 0.125 mg/kg ofPEG20-AQ1-RLX. A single, intravenous dose was administered to eachanimal. N=5 animals per group. MPK: milligrams per kilogram.

FIG. 16A shows a graph of the mean PEG-Relaxin Terminal Half-life versusdose; error bars=SD. FIG. 16B shows a graph of mean PEG-Relaxin AU_(inf)versus dose; error bars=SD.

FIG. 17A shows a graph of the mean PEG-Relaxin Cmax versus dose: errorbars=SD.

FIG. 17B shows a graph of the mean PEG-Relaxin Clearance versus dose;error bars=SD.

FIG. 18A shows a graph of the mean PEG-Relaxin Volume of distributionversus Dose; error bars=SD. FIG. 18B shows a graph of a comparison ofSerum-time concentration after a single intravenous or subcutaneousinjection of 0.25 mg/kg of 20KPEG-AQ1 Relaxin.

FIG. 19A-19F shows data from Phase 1 of Example 43. FIG. 19A shows theeffect of IV infusion with wild-type relaxin on water intake and urineoutput. FIG. 19B shows a baseline for each group of Long-Evans rats from−16 ours to 0 of Phase 1 for urine output. FIG. 19C shows the effect ofIV infusion with wild-type relaxin on hematocrit. FIG. 19D shows theeffect of IV infusion with wild-type relaxin on plasma BUN in the femaleLong-Evans rats. FIG. 19E shows water intake for each group ofLong-Evans rats from 0 to 6 hours of Phase 1. FIG. 19F shows urineoutput for each group of Long-Evans rats from 0 to 6 hours of Phase 1.

FIG. 20A-20I shows data from Phase 2 following administration of vehicle(control group) and test groups with 0.1×, 0.3× and 1× administration ofa 20K PEG-Relaxin variant with A chain substitution in position 1 withpAF bound to PEG (RLX-A-AQ1-20KPEG) from Example 43. FIG. 20A shows theeffect of on water intake and urine output. FIG. 20B shows the effect onplasma sodium levels for each group of Long-Evans rats followinginjection. FIG. 20C shows the effect on plasma sodium change levels foreach group of Long-Evans rats following injection. FIG. 20D shows theeffect of PEG-Relaxin on plasma osmolarity. FIG. 20E shows the effect ofIV infusion with PEG-Relaxin on plasma osmolarity change. FIG. 20F showsthe effect of PEG-Relaxin administration on BUN levels. FIG. 20G showsthe effect of PEG-relaxin administration on water intake for each groupof Long-Evans rats from 0 to 6 hours of Phase 2. FIG. 20H shows baselineurine output for each group of Long-Evans rats from −16 to 0 hours ofPhase 2. FIG. 20I shows the effect of PEG-relaxin administration onurine output for each group of Long-Evans rats from 0 to 6 hours ofPhase 2.

FIGS. 21A-21D show the effect of relaxin in a primary human cardiacfibroblast model of fibrosis. Upregulated expression of collagen type Iand smooth muscle α-actin by human cardiac fibroblasts treated withTGF-β was similarly reduced by treatment with wild-type relaxin orRLX-A-AQ1-20KPEG. Fold changes in gene expression are shown relative tovehicle. Cells were treated with vehicle (Veh), TGF-β (TGF-b) or TGFβtogether with varying amounts of relaxin as indicated, and geneexpression was monitored by PCR. FIGS. 21A and 21C show effect onexpression of collagen type I mRNA. FIGS. 21B and 21D show effect onexpression of smooth muscle α-actin mRNA. Relaxin amounts are expressedrelative to the approximate EC50 value in the CHO-K1 assays usinghRXFP1. For example, 1× indicates 0.1 nM WT-RLX or 3 nMRLX-A-AQ1-20KPEG. Relaxin treatment levels varied between 0.01× and 10×(FIGS. 21A-21B) or between 1× and 100× (FIGS. 21C-21D). Legend:treatment with WT-Relaxin: left bar in each treatment group: treatmentwith RLX-A-AQ1-20KPEG: right bar in each treatment group. Numbers abovebars indicate percent reduction vs. TGF-β only with vehicle set asbaseline. *p<0.05.

FIG. 22A. Rat PK study in which serum levels of RLX-A-AQ1-20KPEG werecompared to changes in serum sodium concentrations. (Left Panel)Measured serum levels of RLX-A-AQ1-20KPEG as related to reported ratpregnancy levels of WT-RLX (˜12 ng/mL) with ˜30-fold adjustment forreduced potency determined in in vitro assays. (Right Panel) Changes inserum sodium concentrations over time with either vehicle (right bar ateach time point) or RLX-A-AQ1-20KPEG treatment (left bar at each timepoint, dosed IV at 0.2 mg/kg). 36 hr sampling not done for vehiclegroup; 0.15 time point substituted for 0.25 time point for vehiclegroup. Means±SEM, *P<0.05 vs. baseline.

FIG. 22B. Rat PK study in which serum levels of RLX-A-AQ1-20KPEG werecompared to changes in serum osmolality. (Left Panel) Measured serumlevels of RLX-A-AQ1-20KPEG as related to reported rat pregnancy levelsof WT-RLX (˜12 ng/mL) with ˜30-fold adjustment for reduced potency in invitro assays. (Right Panel) Changes in serum osmolality over time witheither vehicle or RLX-A-AQ1-20KPEG treatment (dosed as IV bolus at 0.2mg/kg). 36 hr sampling not done for vehicle group; 0.15 time pointsubstituted for 0.25 time point for vehicle group. Means±SEM. No valuesreached statistical significance.

FIG. 23 . Effect of WT-RLX and RLX-A-AQ1-20KPEG on collagen content inthe mouse isoproterenol heart failure model of cardiac fibrosis.Collagen concentration was determined by blinded histological analysisfollowing picrosirius red staining. Significant differences betweenWT-RLX and RLX-A-AQ1-20KPEG and vehicle were determined by One Way ANOVAand Dunnett's test. *P<0.05. Data are mean±SEM.

DETAILED DESCRIPTION Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “relaxin” or “relaxinpolypeptide” and various hyphenated and unhyphenated forms is areference to one or more such proteins and includes equivalents thereofknown to those of ordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

The term “relaxin” as used herein, refers to human relaxin, specificallyhuman relaxin 2 unless the context indicates otherwise, whose amino acidsequence and spatial structure are well-known. Human relaxin iscomprised of a twenty-four amino acid A-chain and a twenty-nine aminoacid B-chain which are cross-linked by disulfide bonds. A properlycross-linked relaxin contains three disulfide bridges: one betweenposition 11 of the A-chain and position 11 of the B-chain, a secondbetween position 24 of the A-chain and position 23 of the B-chain, and athird between positions 10 and 15 of the A-chain.

The terms “wild-type relaxin.” “WT relaxin,” and “wt rhRelaxin” refer toa human relaxin 2 polypeptide having the A chain polypeptide of SEQ IDNO:4 and B chain polypeptide of SEQ ID NO:5 and the disulfide bonds asdepicted in FIG. 4 , wherein position 1 of the A chain is optionallypyroglutamic acid (also known as 5-oxoproline, pidolic acid, pyrrolidonecarboxylic acid, or pyroglutamate for its basic form) which may resultfrom post-translational modification.

The term “RLX-A” refers to a human relaxin 2 polypeptide sequencevariant which differs from the wild-type sequence by the substitution ofan alanine residue (i.e., “A”) for aspartic acid in the first positionof the B chain. By contrast, the terms “RLX-D” or “RLX-D-WT” refer to ahuman relaxin 2 polypeptide having the wild-type residue (aspartic acid,i.e., “D”) at the first position of the B chain.

The terms “RLX-A-AQ1” and “AQ1-RLX” refer to a human relaxin 2 varianthaving alanine at the first position of the B chain wherein the firstposition of the A chain (residue Q1) is substituted with thenon-naturally occurring amino acid para-acetylphenylalanine (pACF),which pACF may be covalently linked to a water soluble polymer such as aPEG, e.g., a 20 kDA PEG. Said pACF may be incorporated using a selectorcodon or using other methods.

The terms “RLX-A-AQ1-20KPEG,” “RLX-A-AQ1-20K,” “PEG20-AQ1-RLX,”“20KPEG-AQ1,” “20KPEG-AQ1-RLX” and “PEG-Relaxin Variant” refer to ahuman relaxin 2 variant having alanine at the first position of the Bchain and wherein the A chain residue Q1 is substituted with thenon-naturally occurring amino acid pACF, which pACF is covalently linkedto a 20 kDA PEG, e.g., through an oxime linkage.

The term “prorelaxin” as used herein is a protein of the formula: B-C-A

wherein:

-   -   A is the A chain of relaxin or a functional derivative thereof;    -   B is the B chain of relaxin or a functional derivative thereof;        and    -   C is the connecting peptide of prorelaxin, wherein the protein        optionally includes one or more of the disulfide bonds of        relaxin. Preferably, prorelaxin is the A chain of human relaxin,        the B chain of human relaxin, and C is the natural connecting        peptide.

The term “relaxin analog” as used herein is a properly cross-linkedprotein exhibiting relaxin activity of the formula: A-B

wherein:

-   -   A is the A chain of relaxin or a functional derivative of the        relaxin A chain; and    -   B is the B chain of relaxin or a functional derivative of the        relaxin B chain and at least one of A or B contains an amino        acid modification from the natural sequence. The hyphen in the        formula “A-B” indicates a physical association of the relaxin A        and B chains but does not necessarily indicate a peptide linkage        between the two chains; rather, the A and B chains may be linked        by one or more disulfide bonds, e.g., as depicted in FIG. 4 .

For example, the term “relaxin analog” includes a protein that has anA-chain and a B-chain that have substantially the same amino acidsequences as the A-chain and/or B-chain of human relaxin, respectively,but differs from the A-chain and/or B-chain of human relaxin by havingone or more amino acid deletions, one or more amino acid replacements,and/or one or more amino acid additions that do not destroy the relaxinactivity of the relaxin analog. A relaxin analog having an isoelectricpoint that is “higher than” the isoelectric point of relaxin is one typeof relaxin analog. Another type of relaxin analog is a “monomericrelaxin analog.”

In the present specification, whenever the term relaxin is used in aplural or a generic sense it is intended to encompass both naturallyoccurring relaxins and relaxin analogues and derivatives thereof.

By “relaxin polypeptide” as used herein is meant a compound having amolecular structure similar to that of human relaxin including thedisulfide bridges between Cys^(A11) and Cys^(B11) and between Cys^(A24)and Cys^(B23) and an internal disulfide bridge between Cys^(A10) andCys^(A15), and which have relaxin activity. Exemplary relaxinpolypeptides include, among others, RLX-A-AQ1 and RLX-A-AQ1-20KPEG.

The term “relaxin polypeptide” also includes the pharmaceuticallyacceptable salts and prodrugs, and prodrugs of the salts, polymorphs,hydrates, solvates, biologically-active fragments, biologically activevariants and stereoisomers of the naturally-occurring relaxin as well asagonist, mimetic, and antagonist variants of the naturally-occurringrelaxin and polypeptide fusions thereof. Fusions comprising additionalamino acids at the amino terminus, carboxyl terminus, or both, areencompassed by the term “relaxin polypeptide.” Exemplary fusionsinclude, but are not limited to, e.g., methionyl relaxin in which amethionine is linked to the N-terminus of relaxin resulting from therecombinant expression of the mature form of relaxin lacking the leaderor signal peptide or portion thereof (a methionine is linked to theN-terminus of relaxin resulting from the recombinant expression),fusions for the purpose of purification (including, but not limited to,to poly-histidine or affinity epitopes), fusions with serum albuminbinding peptides and fusions with serum proteins such as serum albumin.U.S. Pat. No. 5,750,373, which is incorporated by reference herein,describes a method for selecting novel proteins such as growth hormoneand antibody fragment variants having altered binding properties fortheir respective receptor molecules. The method comprises fusing a geneencoding a protein of interest to the carboxy terminal domain of thegene III coat protein of the filamentous phage M13. Chimeric moleculescomprising relaxin and one or more other molecules. The chimericmolecule can contain specific regions or fragments of one or both of therelaxin and the other molecule(s). Any such fragments can be preparedfrom the proteins by standard biochemical methods, or by expressing apolynucleotide encoding the fragment. Relaxin, or a fragment thereof,can be produced as a fusion protein comprising human serum albumin(HSA), Fe, or a portion thereof. Such fusion constructs are suitable forenhancing expression of the relaxin, or fragment thereof, in aneukaryotic host cell. Exemplary HSA portions include the N-terminalpolypeptide (amino acids 1-369, 1-419, and intermediate lengths startingwith amino acid 1), as disclosed in U.S. Pat. No. 5,766,883, andpublication WO 97/24445, which are incorporated by reference herein.Other chimeric polypeptides can include a HSA protein with relaxin, orfragments thereof, attached to each of the C-terminal and N-terminalends of the HSA. Such HSA constructs are disclosed in U.S. Pat. No.5,876,969, which is incorporated by reference herein. Other fusions maybe created by fusion of relaxin with a) the Fc portion of animmunoglobulin: b) an analog of the Fc portion of an immunoglobulin; andc) fragments of the Fc portion of an immunoglobulin.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term “relaxin polypeptide” includespolypeptides conjugated to a polymer such as PEG and may be comprised ofone or more additional derivitizations of cysteine, lysine, or otherresidues. In addition, the relaxin polypeptide may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid according to the presentinvention, or may be conjugated to a naturally encoded amino acidutilizing techniques known in the art such as coupling to lysine orcysteine.

The term “relaxin polypeptide” also includes glycosylated relaxin, suchas but not limited to, polypeptides glycosylated at any amino acidposition. N-linked or O-linked glycosylated forms of the polypeptide.Variants containing single nucleotide changes are also considered asbiologically active variants of relaxin polypeptide. In addition, splicevariants are also included. The term “relaxin polypeptide” also includesrelaxin polypeptide heterodimers, homodimers, heteromultimers, orhomomultimers of any one or more relaxin polypeptides or any otherpolypeptide, protein, carbohydrate, polymer, small molecule, linker,ligand, or other biologically active molecule of any type, linked bychemical means or expressed as a fusion protein, as well as polypeptideanalogues containing, for example, specific deletions or othermodifications yet maintain biological activity.

The term “relaxin polypeptide” or “relaxin” encompasses relaxinpolypeptides comprising one or more amino acid substitutions, additionsor deletions. Relaxin polypeptides of the present invention may becomprised of modifications with one or more natural amino acids inconjunction with one or more non-natural amino acid modification.Exemplary substitutions in a wide variety of amino acid positions innaturally-occurring relaxin polypeptides have been described, includingbut not limited to substitutions that modulate pharmaceutical stability,that modulate one or more of the biological activities of the relaxinpolypeptide, such as but not limited to, increase agonist activity,increase solubility of the polypeptide, decrease proteasesusceptibility, convert the polypeptide into an antagonist, etc. and areencompassed by the term “relaxin polypeptide.” In some embodiments, therelaxin antagonist comprises a non-naturally encoded amino acid linkedto a water soluble polymer that is present in a receptor binding regionof the relaxin molecule.

In some embodiments, the relaxin polypeptides further comprise anaddition, substitution or deletion that modulates biological activity ofthe relaxin polypeptide. For example, the additions, substitutions ordeletions may modulate one or more properties or activities of relaxin.For example, the additions, substitutions or deletions may modulateaffinity for the relaxin receptor, modulate circulating half-life,modulate therapeutic half-life, modulate stability of the polypeptide,modulate cleavage by proteases, modulate dose, modulate release orbio-availability, facilitate purification, or improve or alter aparticular route of administration. Similarly, relaxin polypeptides maycomprise protease cleavage sequences, reactive groups, antibody-bindingdomains (including but not limited to, FLAG or poly-His) or otheraffinity based sequences (including but not limited to, FLAG, poly-His,GST, etc.) or linked molecules (including but not limited to, biotin)that improve detection (including but not limited to, GFP), purificationor other traits of the polypeptide.

The term “relaxin polypeptide” also encompasses homodimers,heterodimers, homomultimers, and heteromultimers that are linked,including but not limited to those linked directly via non-naturallyencoded amino acid side chains, either to the same or differentnon-naturally encoded amino acid side chains, to naturally-encoded aminoacid side chains, or indirectly via a linker. Exemplary linkersincluding but are not limited to, small organic compounds, water solublepolymers of a variety of lengths such as poly(ethylene glycol) orpolydextran, or polypeptides of various lengths.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, para-acetylphenylalanine,N-acetylglucosaminyl-L-serine. N-acetylglucosaminyl-L-threonine, andO-phosphotyrosine.

The term “substantially purified” refers to a relaxin polypeptide thatmay be substantially or essentially free of components that normallyaccompany or interact with the protein as found in its naturallyoccurring environment, i.e. a native cell, or host cell in the case ofrecombinantly produced relaxin polypeptides. Relaxin polypeptide thatmay be substantially free of cellular material includes preparations ofprotein having less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 5%, lessthan about 4%, less than about 3%, less than about 2%, or less thanabout 1% (by dry weight) of contaminating protein. When the relaxinpolypeptide or variant thereof is recombinantly produced by the hostcells, the protein may be present at about 30%, about 25%, about 20%,about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about1% or less of the dry weight of the cells. When the relaxin polypeptideor variant thereof is recombinantly produced by the host cells, theprotein may be present in the culture medium at about 5 g/L, about 4g/L, about 3 g/L, about 2 g/L, about 1 g/L, about 750 mg/L, about 500mg/L, about 250 mg/L, about 100 mg/L, about 50 mg/L, about 10 mg/L, orabout 1 mg/L or less of the dry weight of the cells. Thus,“substantially purified” relaxin polypeptide as produced by the methodsof the present invention may have a purity level of at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, specifically, a purity level of at least about 75%,80%, 85%, and more specifically, a purity level of at least about 90%, apurity level of at least about 95%, a purity level of at least about 99%or greater as determined by appropriate methods such as SDS/PAGEanalysis. RP-HPLC, SEC, and capillary electrophoresis.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassa medium in which the host cell has been grown, e.g., a medium intowhich the relaxin polypeptide has been secreted, including a mediumeither before or after a proliferation step. The term also may encompassbuffers or reagents that contain host cell lysates, such as in the casewhere the relaxin polypeptide is produced intracellularly and the hostcells are lysed or disrupted to release the relaxin polypeptide.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. It is readily apparent tothose of ordinary skill in the art that a wide variety of reducingagents are suitable for use in the methods and compositions of thepresent invention.

“Oxidizing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Itis readily apparent to those of ordinary skill in the art that a widevariety of oxidizing agents are suitable for use in the methods of thepresent invention.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of aprotein. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic solvents, watermiscible solvents, phospholipids, or a combination of two or more suchagents. Suitable chaotropes include, but are not limited to, urea,guanidine, and sodium thiocyanate. Useful detergents may include, butare not limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN→2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),343-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C2-C4 alkanols such as ethanol or isopropanol), orlower alkandiols (especially C2-C4 alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the presentinvention may be naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such ashomoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Reference to an amino acidincludes, for example, naturally occurring proteogenic L-amino acids;D-amino acids, chemically modified amino acids such as amino acidvariants and derivatives; naturally occurring non-proteogenic aminoacids such as β-alanine, ornithine, etc.; and chemically synthesizedcompounds having properties known in the art to be characteristic ofamino acids. Examples of non-naturally occurring amino acids include,but are not limited to, para-acetylphenylalanine, α-methyl amino acids(e.g., α-methyl alanine), D-amino acids, histidine-like amino acids(e.g., 2-amino-histidine, β-hydroxy-histidine, homohistidine,α-fluoromethyl-histidine and α-methyl-histidine), amino acids having anextra methylene in the side chain (“homo” amino acids), and amino acidsin which a carboxylic acid functional group in the side chain isreplaced with a sulfonic acid group (e.g., cysteic acid). Theincorporation of non-natural amino acids, including synthetic non-nativeamino acids, substituted amino acids, or one or more D-amino acids intothe proteins of the present invention may be advantageous in a number ofdifferent ways. D-amino acid-containing peptides, etc., exhibitincreased stability in vitro or in vivo compared to L-aminoacid-containing counterparts. Thus, the construction of peptides, etc.,incorporating D-amino acids can be particularly useful when greaterintracellular stability is desired or required. More specifically,D-peptides, etc., are resistant to endogenous peptidases and proteases,thereby providing improved bioavailability of the molecule, andprolonged lifetimes in vivo when such properties are desirable.Additionally, D-peptides, etc., cannot be processed efficiently formajor histocompatibility complex class IT-restricted presentation to Thelper cells, and are therefore, less likely to induce humoral immuneresponses in the whole organism.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences. “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.: 2nd edition (December 1993).

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

The terms “functional group” “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde: acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5 hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs,vaccines, immunogens, hard drugs, soft drugs, carbohydrates, inorganicatoms or molecules, dyes, lipids, nucleosides, radionuclides,oligonucleotides, toxoids, toxins, prokaryotic and eukaryotic cells,viruses, polysaccharides, nucleic acids and portions thereof obtained orderived from viruses, bacteria, insects, animals or any other cell orcell type, liposomes, microparticles and micelles. The relaxinpolypeptides may be added in a micellular formulation; see U.S. Pat. No.5,833,948, which is incorporated by reference herein in its entirety.Classes of biologically active agents that are suitable for use with theinvention include, but are not limited to, drugs, prodrugs,radionuclides, imaging agents, polymers, antibiotics, fungicides,anti-viral agents, anti-inflammatory agents, anti-tumor agents,cardiovascular agents, anti-anxiety agents, hormones, growth factors,steroidal agents, microbially derived toxins, and the like.

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789which are incorporated by reference herein. A “multi-functional polymer”refers to a polymer comprising two or more discrete functional groupsthat are capable of reacting specifically with other moieties (includingbut not limited to, amino acid side groups) to form covalent ornon-covalent linkages. A bi-functional polymer or multi-functionalpolymer may be any desired length or molecular weight, and may beselected to provide a particular desired spacing or conformation betweenone or more molecules linked to the relaxin and its receptor or relaxin.

Where substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, the structure CH2O isequivalent to the structure —OCH2.

The term “substituents” includes but is not limited to “non-interferingsubstituents”. “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C1-C10 alkyl, C2-C10alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C1-C12 aralkyl, C1-C12 alkaryl,C3-C12 cycloalkyl, C3-C12 cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, C2-C12 alkoxyaryl,C7-C12 aryloxyalkyl, C7-C12 oxyaryl, C1-C6 alkylsulfinyl, C1-C10alkylsulfonyl, —(CH2)m-O—(C1-C10 alkyl) wherein m is from 1 to 8, aryl,substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical,substituted heterocyclic radical, nitroalkyl, —NO2, —CN, —NRC(O)—(C1-C10alkyl), —C(O)—(C1-C10 alkyl), C2-C10 alkyl thioalkyl, —C(O)O—(C1-C10alkyl), —OH, —SO2, ═S, —COOH, —NR2, carbonyl, —C(O)—(C1-C10 alkyl)-CF3,—C(O)—CF3, —C(O)NR2, —(C1-C10 aryl)-S—(C6-C10 aryl), —C(O)—(C1-C10aryl), —(CH2)m-O—(—(CH2)m-O—(C1-C10 alkyl) wherein each m is from 1 to8, —C(O)NR2, —C(S)NR2, —SO2NR2, —NRC(O) NR2, —NRC(S) NR2, salts thereof,and the like. Each R as used herein is H, alkyl or substituted alkyl,aryl or substituted aryl, aralkyl, or alkaryl.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C1-C10means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by the structures —CH2CH2- and —CH2CH2CH2CH2-, and furtherincludes those groups described below as “heteroalkylene.” Typically, analkyl (or alkylene) group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being a particularembodiment of the methods and compositions described herein. A “loweralkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH2-CH2-O—CH3,—CH2-CH2-NH—CH3, —CH2-CH2-N(CH3)-CH3, —CH2-S—CH2-CH3, —CH2-CH2,—S(O)—CH3, —CH2-CH2-S(O)2-CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2-CH═N—OCH3,and —CH═CH—N(CH3)-CH3. Up to two heteroatoms may be consecutive, suchas, for example, —CH2-NH—OCH3 and —CH2-O—Si(CH3)3. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH2-CH2-S—CH2 CH2- and —CH2-S—CH2-CH2-NH—CH2-. Forheteroalkylene groups, the same or different heteroatoms can also occupyeither or both of the chain termini (including but not limited to,alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,aminooxyalkylene, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)2R′ represents both —C(O)2R′ and—R′C(O)2.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated, partially unsaturated and fullyunsaturated ring linkages. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like. Additionally, the term encompasses bicyclicand tricyclic ring structures. Similarly, the term “heterocycloalkylene”by itself or as part of another substituent means a divalent radicalderived from heterocycloalkyl, and the term “cycloalkylene” by itself oras part of another substituent means a divalent radical derived fromcycloalkyl.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto relaxin polypeptides can result in changes including, but not limitedto, increased or modulated serum half-life, or increased or modulatedtherapeutic half-life relative to the unmodified form, modulatedimmunogenicity, modulated physical association characteristics such asaggregation and multimer formation, altered receptor binding, alteredbinding to one or more binding partners, and altered receptordimerization or multimerization. The water soluble polymer may or maynot have its own biological activity, and may be utilized as a linkerfor attaching relaxin to other substances, including but not limited toone or more relaxin polypeptides, or one or more biologically activemolecules. Suitable polymers include, but are not limited to,polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C10alkoxy or aryloxy derivatives thereof (described in U.S. Pat. No.5,252,714 which is incorporated by reference herein),monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinylalcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (poly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” encompasses both linear and branched polymers andaverage molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (including but not limited to, from 1 to 3 rings) which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from one to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a heteroatom. Non-limiting examples of aryl and heteroarylgroups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(including but not limited to, aryloxy, arylthioxy, arylalkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the term“arylalkyl” is meant to include those radicals in which an aryl group isattached to an alkyl group (including but not limited to, benzyl,phenethyl, pyridylmethyl and the like) including those alkyl groups inwhich a carbon atom (including but not limited to, a methylene group)has been replaced by, for example, an oxygen atom (including but notlimited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,and the like).

Each of the above terms (including but not limited to, “alkyl,”“heteroalkyl,” “aryl” and “heteroaryl”) are meant to include bothsubstituted and unsubstituted forms of the indicated radical. Exemplarysubstituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, OC(O)R′, —C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, NR″C(O)NR—R′″, —NR″C(O)2R′, —NR—C(NR′R″R′″)═NR″″, NRC(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, NRSO2R′, —CN and —NO2 ina number ranging from zero to (2m′+1), where m′ is the total number ofcarbon atoms in such a radical. R′, R″, R′″ and R″″ each independentlyrefer to hydrogen, substituted or unsubstituted heteroalkyl, substitutedor unsubstituted aryl, including but not limited to, aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, I-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (including butnot limited to, —CF3 and —CH2CF3) and acyl (including but not limitedto, —C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, but are not limited to: halogen, OR′, ═O, ═NR′, ═N—OR′,—NR′R′, —SR′, -halogen, —SiR′R″R′″, OC(O)R′, —C(O)R′, CO2R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, NR′ C(O)NR″R′″, —NR″C(O)2R′,NR—C(NR′R″R″)═NR′″, NR C(NR′R′)═NR′, —S(O)R′, —S(O)2R′, —S(O)2NR′R″,NRSO2R′, —CN and —NO2, —R′, —N3, —CH(Ph)2, fluoro(C1-C4)alkoxy, andfluoro(C1-C4)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R %, R″, R′″ andR″″ are independently selected from hydrogen, alkyl, heteroalkyl, aryland heteroaryl. When a compound of the invention includes more than oneR group, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified relaxinrelative to its non-modified form. Serum half-life is measured by takingblood samples at various time points after administration of relaxin,and determining the concentration of that molecule in each sample.Correlation of the serum concentration with time allows calculation ofthe serum half-life. Increased serum half-life desirably has at leastabout two-fold, but a smaller increase may be useful, for example whereit enables a satisfactory dosing regimen or avoids a toxic effect. Insome embodiments, the increase is at least about three-fold, at leastabout five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of relaxin, relative to its non-modified form.Therapeutic half-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, increased ordecreased breakdown of the molecule by enzymes such as proteases, or anincrease or decrease in another parameter or mechanism of action of thenon-modified molecule or an increase or decrease in receptor-mediatedclearance of the molecule.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms (or other algorithms available to persons of ordinary skillin the art) or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence of a polynucleotide or polypeptide. A polynucleotide encoding apolypeptide of the present invention, including homologs from speciesother than human, may be obtained by a process comprising the steps ofscreening a library under stringent hybridization conditions with alabeled probe having a polynucleotide sequence of the invention or afragment thereof, and isolating full-length cDNA and genomic clonescontaining said polynucleotide sequence. Such hybridization techniquesare well known to the skilled artisan.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are known to those of ordinary skill in the art. Optimalalignment of sequences for comparison can be conducted, including butnot limited to, by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, WI), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationavailable at the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithmparameters W, T, and X determine the sensitivity and speed of thealignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm istypically performed with the “low complexity” filter turned off.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, or less than about0.01, or less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, PNA, or other nucleic acid mimics, orcombinations thereof under conditions of low ionic strength and hightemperature as is known in the art. Typically, under stringentconditions a probe will hybridize to its target subsequence in a complexmixture of nucleic acid (including but not limited to, total cellular orlibrary DNA or RNA) but does not hybridize to other sequences in thecomplex mixture. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(Tm) for the specific sequence at a defined ionic strength and pH. TheTm is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (including butnot limited to, 10 to 50 nucleotides) and at least about 60° C. for longprobes (including but not limited to, greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. For selective or specifichybridization, a positive signal may be at least two times background,optionally 10 times background hybridization. Exemplary stringenthybridization conditions can be as following: 50% formamide, 5×SSC, and1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C.with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can beperformed for 5, 15, 30, 60, 120, or more minutes.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “non-eukaryote” refers to non-cukaryoticorganisms. For example, a non-eukaryotic organism can belong to theEubacteria (including but not limited to, Escherichia coli, Thermusthermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain,or the Archaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment. An animal may be acompanion animal (e.g., dogs, cats, and the like), farm animal (e.g.,cows, sheep, pigs, horses, and the like) or a laboratory animal (e.g.,rats, mice, guinea pigs, and the like).

The term “effective amount” as used herein refers to that amount of themodified non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing themodified non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein, includingphotolabile groups such as Nvoc and McNvoc. Other protecting groupsknown in the art may also be used in or with the methods andcompositions described herein.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

The terms “disorders of the cardiovascular system” or “cardiovasculardisorders” include for example the following disorders: hypertension(high blood pressure), peripheral and cardiac vascular disorders,coronary heart disease, stable and unstable angina pectoris, myocardialinsufficiency, persistent ischemic dysfunction (“hibernatingmyocardium”), temporary postischemic dysfunction (“stunned myocardium”),heart failure, disturbances of peripheral blood flow, acute coronarysyndrome, heart failure and myocardial infarction.

The term “heart failure” includes both acute and chronic manifestationsof heart failure, as well as more specific or related types of disease,such as acute decompensated heart failure, right heart failure, leftheart failure, global failure, ischemic cardiomyopathy, dilatedcardiomyopathy, congenital heart defects, heart valve defects, heartfailure associated with heart valve defects, mitral stenosis, mitralinsufficiency, aortic stenosis, aortic insufficiency, tricuspidstenosis, tricuspid insufficiency, pulmonary stenosis, pulmonary valveinsufficiency, combined heart valve defects, myocardial inflammation(myocarditis), chronic myocarditis, acute myocarditis, viralmyocarditis, diabetic heart failure, alcoholic cardiomyopathy, cardiacstorage disorders, cardiomyopathy of infection, toxic cardiomyopathy(e.g., associated with use of chemotherapeutic agents, cocaine, ormethamphetamine), heart failure associated with arrhythmia, heartfailure associated with diabetes, heart failure associated with severeanemia, heart failure associated with hyperthyroidism, heart failureassociated with hypothyroidism, heart failure associated with emphysema,heart failure associated with lupus, heart failure associated withhemochromatosis, heart failure associated with amyloidosis, heartfailure associated with sarcoidosis, heart failure associated withinfection (e.g., viral infection), heart failure associated withallergic reactions, heart failure associated with blood clots in thelungs, heart failure associated with medications, or other heart failureassociated with an illness (e.g., a severe illness that affects thewhole body) and diastolic and systolic heart failure and acute phases ofworsening heart failure.

The term “fibrotic disorders” encompasses diseases and disorderscharacterized by fibrosis, including among others the following diseasesand disorders: hepatic fibrosis, cirrhosis of the liver, pulmonaryfibrosis, endomyocardial fibrosis, nephropathy, polycystic kidneydisease, glomerulonephritis, interstitial renal fibrosis, fibroticdamage resulting from diabetes, bone marrow fibrosis and similarfibrotic disorders, scleroderma, morphea, keloids, hypertrophic scarring(also following surgical procedures), naevi, diabetic retinopathy,proliferative vitreoretinopathy and disorders of the connective tissue(for example sarcoidosis).

Non-naturally encoded amino acid polypeptides presented herein mayinclude isotopically-labelled compounds with one or more atoms replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as 2H,3H, 13C, 14C, 15N, 18O, 17O, 35S, 18F, 36Cl, respectively. Certainisotopically-labelled compounds described herein, for example those intowhich radioactive isotopes such as 3H and 14C are incorporated, may beuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., 2H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

All isomers including but not limited to diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein. In additional or further embodiments, the non-naturally encodedamino acid polypeptides are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-naturally encodedamino acid polypeptides.

In some situations, non-naturally encoded amino acid polypeptides mayexist as tautomers. In addition, the non-naturally encoded amino acidpolypeptides described herein can exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water,ethanol, and the like. The solvated forms are also considered to bedisclosed herein. Those of ordinary skill in the art will recognize thatsome of the compounds herein can exist in several tautomeric forms. Allsuch tautomeric forms are considered as part of the compositionsdescribed herein.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

I. INTRODUCTION

Relaxin polypeptides comprising at least one unnatural amino acid areprovided in the invention. In certain embodiments of the invention, therelaxin polypeptide with at least one unnatural amino acid includes atleast one post-translational modification. In one embodiment, the atleast one post-translational modification comprises attachment of amolecule including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove or any other desirable compound or substance, comprising a secondreactive group to at least one unnatural amino acid comprising a firstreactive group utilizing chemistry methodology that is known to one ofordinary skill in the art to be suitable for the particular reactivegroups. For example, the first reactive group is an alkynyl moiety(including but not limited to, in the unnatural amino acidp-propargyloxyphenylalanine, where the propargyl group is also sometimesreferred to as an acetylene moiety) and the second reactive group is anazido moiety, and [3+2] cycloaddition chemistry methodologies areutilized. In another example, the first reactive group is the azidomoiety (including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety. In certain embodiments of the modified relaxin polypeptide ofthe present invention, at least one unnatural amino acid (including butnot limited to, unnatural amino acid containing a keto functional group)comprising at least one post-translational modification, is used wherethe at least one post-translational modification comprises a saccharidemoiety. In certain embodiments, the post-translational modification ismade in vivo in a eukaryotic cell or in a non-eukaryotic cell. A linker,polymer, water soluble polymer, or other molecule may attach themolecule to the polypeptide. The molecule may be linked directly to thepolypeptide.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike.

In some embodiments, the relaxin polypeptide comprises one or morenon-naturally encoded amino acids for glycosylation, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, or glycolipid-linkage modification of the polypeptide.In some embodiments, the relaxin polypeptide comprises one or morenon-naturally encoded amino acids for glycosylation of the polypeptide.In some embodiments, the relaxin polypeptide comprises one or morenaturally encoded amino acids for glycosylation, acetylation, acylation,lipid-modification, palmitoylation, palmitate addition, phosphorylation,or glycolipid-linkage modification of the polypeptide. In someembodiments, the relaxin polypeptide comprises one or more naturallyencoded amino acids for glycosylation of the polypeptide.

In some embodiments, the relaxin polypeptide comprises one or morenon-naturally encoded amino acid additions and/or substitutions thatenhance glycosylation of the polypeptide. In some embodiments, therelaxin polypeptide comprises one or more deletions that enhanceglycosylation of the polypeptide. In some embodiments, the relaxinpolypeptide comprises one or more non-naturally encoded amino acidadditions and/or substitutions that enhance glycosylation at a differentamino acid in the polypeptide. In some embodiments, the relaxinpolypeptide comprises one or more deletions that enhance glycosylationat a different amino acid in the polypeptide. In some embodiments, therelaxin polypeptide comprises one or more non-naturally encoded aminoacid additions and/or substitutions that enhance glycosylation at anon-naturally encoded amino acid in the polypeptide. In someembodiments, the relaxin polypeptide comprises one or more non-naturallyencoded amino acid additions and/or substitutions that enhanceglycosylation at a naturally encoded amino acid in the polypeptide. Insome embodiments, the relaxin polypeptide comprises one or morenaturally encoded amino acid additions and/or substitutions that enhanceglycosylation at a different amino acid in the polypeptide. In someembodiments, the relaxin polypeptide comprises one or more non-naturallyencoded amino acid additions and/or substitutions that enhanceglycosylation at a naturally encoded amino acid in the polypeptide. Insome embodiments, the relaxin polypeptide comprises one or morenon-naturally encoded amino acid additions and/or substitutions thatenhance glycosylation at a non-naturally encoded amino acid in thepolypeptide.

In one embodiment, the post-translational modification comprisesattachment of an oligosaccharide to an asparagine by a GlcNAc-asparaginelinkage (including but not limited to, where the oligosaccharidecomprises (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, and the like). In anotherembodiment, the post-translational modification comprises attachment ofan oligosaccharide (including but not limited to, Gal-GalNAc,Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine, aGalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. Incertain embodiments, a protein or polypeptide of the invention cancomprise a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, and/or the like. Examples ofsecretion signal sequences include, but are not limited to, aprokaryotic secretion signal sequence, a eukaryotic secretion signalsequence, a eukaryotic secretion signal sequence 5′-optimized forbacterial expression, a novel secretion signal sequence, pectate lyasesecretion signal sequence, Omp A secretion signal sequence, and a phagesecretion signal sequence. Examples of secretion signal sequences,include, but are not limited to, STII (prokaryotic), Fd GIII and M13(phage), Bgl2 (yeast), and the signal sequence bla derived from atransposon. Any such sequence may be modified to provide a desiredresult with the polypeptide, including but not limited to, substitutingone signal sequence with a different signal sequence, substituting aleader sequence with a different leader sequence, etc.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

The present invention provides methods and compositions based on relaxincomprising at least one non-naturally encoded amino acid. Introductionof at least one non-naturally encoded amino acid into relaxin can allowfor the application of conjugation chemistries that involve specificchemical reactions, including, but not limited to, with one or morenon-naturally encoded amino acids while not reacting with the commonlyoccurring 20 amino acids. In some embodiments, relaxin comprising thenon-naturally encoded amino acid is linked to a water soluble polymer,such as polyethylene glycol (PEG), via the side chain of thenon-naturally encoded amino acid. This invention provides a highlyefficient method for the selective modification of proteins with PEGderivatives, which involves the selective incorporation ofnon-genetically encoded amino acids, including but not limited to, thoseamino acids containing functional groups or substituents not found inthe 20 naturally incorporated amino acids, including but not limited toa ketone, an azide or acetylene moiety, into proteins in response to aselector codon and the subsequent modification of those amino acids witha suitably reactive PEG derivative. Once incorporated, the amino acidside chains can then be modified by utilizing chemistry methodologiesknown to those of ordinary skill in the art to be suitable for theparticular functional groups or substituents present in thenon-naturally encoded amino acid. Known chemistry methodologies of awide variety are suitable for use in the present invention toincorporate a water soluble polymer into the protein. Such methodologiesinclude but are not limited to a Huisgen [3+2]cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, acetylene or azidederivatives, respectively.

Because the Huisgen [3+2] cycloaddition method involves a cycloadditionrather than a nucleophilic substitution reaction, proteins can bemodified with extremely high selectivity. The reaction can be carriedout at room temperature in aqueous conditions with excellentregioselectivity (1,4>1,5) by the addition of catalytic amounts of Cu(1)salts to the reaction mixture. See, e.g., Tornoe, et al., (2002) J. Org.Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int.Ed. 41:2596-2599; and WO 03/101972. A molecule that can be added to aprotein of the invention through a [3+2] cycloaddition includesvirtually any molecule with a suitable functional group or substituentincluding but not limited to an azido or acetylene derivative. Thesemolecules can be added to an unnatural amino acid with an acetylenegroup, including but not limited to, p-propargyloxyphenylalanine, orazido group, including but not limited to p-azido-phenylalanine,respectively.

The five-membered ring that results from the Huisgen [3+2] cycloadditionis not generally reversible in reducing environments and is stableagainst hydrolysis for extended periods in aqueous environments.Consequently, the physical and chemical characteristics of a widevariety of substances can be modified under demanding aqueous conditionswith the active PEG derivatives of the present invention. Even moreimportantly, because the azide and acetylene moieties are specific forone another (and do not, for example, react with any of the 20 common,genetically-encoded amino acids), proteins can be modified in one ormore specific sites with extremely high selectivity.

The invention also provides water soluble and hydrolytically stablederivatives of PEG derivatives and related hydrophilic polymers havingone or more acetylene or azide moieties. The PEG polymer derivativesthat contain acetylene moieties are highly selective for coupling withazide moieties that have been introduced selectively into proteins inresponse to a selector codon. Similarly, PEG polymer derivatives thatcontain azide moieties are highly selective for coupling with acetylenemoieties that have been introduced selectively into proteins in responseto a selector codon.

More specifically, the azide moieties comprise, but are not limited to,alkyl azides, aryl azides and derivatives of these azides. Thederivatives of the alkyl and aryl azides can include other substituentsso long as the acetylene-specific reactivity is maintained. Theacetylene moieties comprise alkyl and aryl acetylenes and derivatives ofeach. The derivatives of the alkyl and aryl acetylenes can include othersubstituents so long as the azide-specific reactivity is maintained.

The present invention provides conjugates of substances having a widevariety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin;an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; an actinicradiation excitable moiety; a photoisomerizable moiety; biotin; aderivative of biotin; a biotin analogue; a moiety incorporating a heavyatom; a chemically cleavable group; a photocleavable group; an elongatedside chain; a carbon-linked sugar; a redox-active agent; an aminothioacid; a toxic moiety; an isotopically labeled moiety; a biophysicalprobe; a phosphorescent group; a chemiluminescent group: an electrondense group: a magnetic group; an intercalating group; a chromophore; anenergy transfer agent; a biologically active agent; a detectable label;a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; aradiotransmitter; a neutron-capture agent; or any combination of theabove, or any other desirable compound or substance. The presentinvention also includes conjugates of substances having azide oracetylene moieties with PEG polymer derivatives having the correspondingacetylene or azide moieties. For example, a PEG polymer containing anazide moiety can be coupled to a biologically active molecule at aposition in the protein that contains a non-genetically encoded aminoacid bearing an acetylene functionality. The linkage by which the PEGand the biologically active molecule are coupled includes but is notlimited to the Huisgen [3+2] cycloaddition product.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar,R., J. Pharm Pharm Sci., 3(1):125-136 (2000) which are incorporated byreference herein). The invention also includes biomaterials comprising asurface having one or more reactive azide or acetylene sites and one ormore of the azide- or acetylene-containing polymers of the inventioncoupled to the surface via the Huisgen [3+2] cycloaddition linkage.Biomaterials and other substances can also be coupled to the azide- oracetylene-activated polymer derivatives through a linkage other than theazide or acetylene linkage, such as through a linkage comprising acarboxylic acid, amine, alcohol or thiol moiety, to leave the azide oracetylene moiety available for subsequent reactions.

The invention includes a method of synthesizing the azide- andacetylene-containing polymers of the invention. In the case of theazide-containing PEG derivative, the azide can be bonded directly to acarbon atom of the polymer. Alternatively, the azide-containing PEGderivative can be prepared by attaching a linking agent that has theazide moiety at one terminus to a conventional activated polymer so thatthe resulting polymer has the azide moiety at its terminus. In the caseof the acetylene-containing PEG derivative, the acetylene can be bondeddirectly to a carbon atom of the polymer. Alternatively, theacetylene-containing PEG derivative can be prepared by attaching alinking agent that has the acetylene moiety at one terminus to aconventional activated polymer so that the resulting polymer has theacetylene moiety at its terminus.

More specifically, in the case of the azide-containing PEG derivative, awater soluble polymer having at least one active hydroxyl moietyundergoes a reaction to produce a substituted polymer having a morereactive moiety, such as a mesylate, tresylate, tosylate or halogenleaving group, thereon. The preparation and use of PEG derivativescontaining sulfonyl acid halides, halogen atoms and other leaving groupsare known to those of ordinary skill in the art. The resultingsubstituted polymer then undergoes a reaction to substitute for the morereactive moiety an azide moiety at the terminus of the polymer.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an azide at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the azidemoiety is positioned at the terminus of the polymer. Nucleophilic andelectrophilic moieties, including amines, thiols, hydrazides,hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters andthe like, are known to those of ordinary skill in the art.

More specifically, in the case of the acetylene-containing PEGderivative, a water soluble polymer having at least one active hydroxylmoiety undergoes a reaction to displace a halogen or other activatedleaving group from a precursor that contains an acetylene moiety.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an acetylene at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the acetylenemoiety is positioned at the terminus of the polymer. The use of halogenmoieties, activated leaving group, nucleophilic and electrophilicmoieties in the context of organic synthesis and the preparation and useof PEG derivatives is well established to practitioners in the art.

The invention also provides a method for the selective modification ofproteins to add other substances to the modified protein, including butnot limited to water soluble polymers such as PEG and PEG derivativescontaining an azide or acetylene moiety. The azide- andacetylene-containing PEG derivatives can be used to modify theproperties of surfaces and molecules where biocompatibility, stability,solubility and lack of immunogenicity are important, while at the sametime providing a more selective means of attaching the PEG derivativesto proteins than was previously known in the art.

General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, nucleic acids encodinga relaxin polypeptide of interest will be isolated, cloned and oftenaltered using recombinant methods. Such embodiments are used, includingbut not limited to, for protein expression or during the generation ofvariants, derivatives, expression cassettes, or other sequences derivedfrom a relaxin polypeptide. In some embodiments, the sequences encodingthe polypeptides of the invention are operably linked to a heterologouspromoter.

A nucleotide sequence encoding a relaxin polypeptide comprising anon-naturally encoded amino acid may be synthesized on the basis of theamino acid sequence of the parent polypeptide, including but not limitedto, having the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO:2 and then changing the nucleotide sequence so as to effect introduction(i.e., incorporation or substitution) or removal (i.e., deletion orsubstitution) of the relevant amino acid residue(s). The nucleotidesequence may be conveniently modified by site-directed mutagenesis inaccordance with conventional methods. Alternatively, the nucleotidesequence may be prepared by chemical synthesis, including but notlimited to, by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990), and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel. Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, CA (Berger);Sambrook et al., Molecular Cloning A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes or polynucleotides thatinclude selector codons for production of proteins that includeunnatural amino acids, orthogonal tRNAs, orthogonal synthetases, andpairs thereof.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce novel synthetases ortRNAs, to mutate tRNA molecules, to mutate polynucleotides encodingsynthetases, to produce libraries of tRNAs, to produce libraries ofsynthetases, to produce selector codons, to insert selector codons thatencode unnatural amino acids in a protein or polypeptide of interest.They include but are not limited to site-directed, random pointmutagenesis, homologous recombination, DNA shuffling or other recursivemutagenesis methods, chimeric construction, mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like, PCT-mediated mutagenesis, or any combinationthereof. Additional suitable methods include point mismatch repair,mutagenesis using repair-deficient host strains, restriction-selectionand restriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the present invention. In one embodiment, mutagenesis can beguided by known information of the naturally occurring molecule oraltered or mutated naturally occurring molecule, including but notlimited to, sequence, sequence comparisons, physical properties,secondary, tertiary, or quaternary structure, crystal structure or thelike.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis. Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment. Nucleic Acids Res.10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods inEnzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987):Taylor et al., The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764(1985): Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Sayers et al., 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985): Grundströmet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA. 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, 1. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of synthetases, or altering tRNAs, aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes., 12:6159-6168 (1984).

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNA/RS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the invention or constructs which include apolynucleotide of the invention, including but not limited to, a vectorof the invention, which can be, for example, a cloning vector or anexpression vector. For example, the coding regions for the orthogonaltRNA, the orthogonal tRNA synthetase, and the protein to be derivatizedare operably linked to gene expression control elements that arefunctional in the desired host cell. The vector can be, for example, inthe form of a plasmid, a cosmid, a phage, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985)), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)), and/or the like. Techniques suitable for thetransfer of nucleic acid into cells in vitro include the use ofliposomes, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. In vivo gene transfer techniquesinclude, but are not limited to, transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection [Dzau et al., Trends in Biotechnology 11:205-210 (1993)].In some situations it may be desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81(1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al.,Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (allsupra). A catalogue of bacteria and bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, NY. In addition, essentially any nucleic acid (andvirtually any labeled nucleic acid, whether standard or non-standard)can be custom or standard ordered from any of a variety of commercialsources, such as the Midland Certified Reagent Company (Midland, TXavailable on the World Wide Web at mcrc.com), The Great American GeneCompany (Ramona, CA available on the World Wide Web at genco.com),ExpressGen Inc. (Chicago, IL available on the World Wide Web atexpressgen.com), Operon Technologies Inc. (Alameda, CA) and many others.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), anochre codon, or an opal codon (UGA), an unnatural codon, a four or morebase codon, a rare codon, or the like. It is readily apparent to thoseof ordinary skill in the art that there is a wide range in the number ofselector codons that can be introduced into a desired gene orpolynucleotide, including but not limited to, one or more, two or more,three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotideencoding at least a portion of the relaxin polypeptide. It is alsoreadily apparent to those of ordinary skill in the art that there is awide range in the number of selector codons that can be introduced intoa desired gene or polynucleotide, including but not limited to, one ormore, two or more, three or more, 4, 5, 6, 7, 8, 9, 10 or more totalfound in the A chain and B chain polynucleotide sequences encoding atleast a portion of the relaxin polypeptide.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of one or more unnatural aminoacids in vivo. For example, an O-tRNA is produced that recognizes thestop codon, including but not limited to, UAG, and is aminoacylated byan O—RS with a desired unnatural amino acid. This O-tRNA is notrecognized by the naturally occurring host's aminoacyl-tRNA synthetases.Conventional site-directed mutagenesis can be used to introduce the stopcodon, including but not limited to, TAG, at the site of interest in apolypeptide of interest. See, e.g., Sayers, J. R., et al. (1988), 5′-3′Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res, 16:791-802. When the O—RS, O-tRNA andthe nucleic acid that encodes the polypeptide of interest are combinedin vivo, the unnatural amino acid is incorporated in response to the UAGcodon to give a polypeptide containing the unnatural amino acid at thespecified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurring tRNAArg,which exists as a minor species in Escherichia coli. Some organisms donot use all triplet codons. An unassigned codon AGA in Micrococcusluteus has been utilized for insertion of amino acids in an in vitrotranscription/translation extract. See, e.g., Kowal and Oliver, Nucl.Acid. Res., 25:4685 (1997). Components of the present invention can begenerated to use these rare codons in vivo.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, but are not limited to,AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, including but not limited to, one or multiple unnatural aminoacids into the same protein. For example, in the presence of mutatedO-tRNAs, including but not limited to, a special frameshift suppressortRNAs, with anticodon loops, for example, with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, includingbut not limited to, at least a four-base codon, at least a five-basecodon, or at least a six-base codon or more. Since there are 256possible four-base codons, multiple unnatural amino acids can be encodedin the same cell using a four or more base codon. See, Anderson et al.,(2002) Exploring the Limits of Codon and Anticodon Size, Chemistry andBiology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry. 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in the present invention,which can reduce missense readthrough and frameshift suppression atother unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., etal., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (199%) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274.A 3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., Tac et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods known to one of ordinary skill in the art and describedherein to include, for example, one or more selector codon for theincorporation of an unnatural amino acid. For example, a nucleic acidfor a protein of interest is mutagenized to include one or more selectorcodon, providing for the incorporation of one or more unnatural aminoacids. The invention includes any such variant, including but notlimited to, mutant, versions of any protein, for example, including atleast one unnatural amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more unnatural amino acid.

Nucleic acid molecules encoding a protein of interest such as a relaxinpolypeptide may be readily mutated to introduce a cysteine at anydesired position of the polypeptide. Cysteine is widely used tointroduce reactive molecules, water soluble polymers, proteins, or awide variety of other molecules, onto a protein of interest. Methodssuitable for the incorporation of cysteine into a desired position of apolypeptide are known to those of ordinary skill in the art, such asthose described in U.S. Pat. No. 6,608,183, which is incorporated byreference herein, and standard mutagenesis techniques.

Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into a relaxin polypeptide. In general,the introduced non-naturally encoded amino acids are substantiallychemically inert toward the 20 common, genetically-encoded amino acids(i.e., alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine). In some embodiments, thenon-naturally encoded amino acids include side chain functional groupsthat react efficiently and selectively with functional groups not foundin the 20 common amino acids (including but not limited to, azido,ketone, aldehyde and aminooxy groups) to form stable conjugates. Forexample, a relaxin polypeptide that includes a non-naturally encodedamino acid containing an azido functional group can be reacted with apolymer (including but not limited to, poly(ethylene glycol) or,alternatively, a second polypeptide containing an alkyne moiety to forma stable conjugate resulting for the selective reaction of the azide andthe alkyne functional groups to form a Huisgen [3+2] cycloadditionproduct.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the present invention. Because the non-naturally encoded amino acidsof the invention typically differ from the natural amino acids only inthe structure of the side chain, the non-naturally encoded amino acidsform amide bonds with other amino acids, including but not limited to,natural or non-naturally encoded, in the same manner in which they areformed in naturally occurring polypeptides.

However, the non-naturally encoded amino acids have side chain groupsthat distinguish them from the natural amino acids. For example, Roptionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-,hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol,seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine,amino group, or the like or any combination thereof. Other non-naturallyoccurring amino acids of interest that may be suitable for use in thepresent invention include, but are not limited to, amino acidscomprising a photoactivatable cross-linker, spin-labeled amino acids,fluorescent amino acids, metal binding amino acids, metal-containingamino acids, radioactive amino acids, amino acids with novel functionalgroups, amino acids that covalently or noncovalently interact with othermolecules, photocaged and/or photoisomerizable amino acids, amino acidscomprising biotin or a biotin analogue, glycosylated amino acids such asa sugar substituted serine, other carbohydrate modified amino acids,keto-containing amino acids, amino acids comprising polyethylene glycolor polyether, heavy atom substituted amino acids, chemically cleavableand/or photocleavable amino acids, amino acids with an elongated sidechains as compared to natural amino acids, including but not limited to,polyethers or long chain hydrocarbons, including but not limited to,greater than about 5 or greater than about 10 carbons, carbon-linkedsugar-containing amino acids, redox-active amino acids, amino thioacidcontaining amino acids, and amino acids comprising one or more toxicmoiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, MO, USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, MA, USA). Those that are not commercially availableare optionally synthesized as provided herein or using standard methodsknown to those of ordinary skill in the art. For organic synthesistechniques, see, e.g., Organic Chemistry by Fessendon and Fessendon,(1982, Second Edition, Willard Grant Press, Boston Mass.); AdvancedOrganic Chemistry by March (Third Edition, 1985, Wiley and Sons, NewYork); and Advanced Organic Chemistry by Carey and Sundberg (ThirdEdition, Parts A and B, 1990, Plenum Press, New York). See, also, U.S.Pat. Nos. 7,045,337 and 7,083,970, which are incorporated by referenceherein. In addition to unnatural amino acids that contain novel sidechains, unnatural amino acids that may be suitable for use in thepresent invention also optionally comprise modified backbone structures,including but not limited to, as illustrated by the structures ofFormula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C 6-C20 straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, a-hydroxy derivatives, g-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcb-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, which is incorporated by reference herein, foradditional methionine analogs. International Application No.PCT/US06/47822 entitled “Compositions Containing, Methods Involving, andUses of Non-natural Amino Acids and Polypeptides,” which is incorporatedby reference herein, describes reductive alkylation of an aromatic aminemoieties, including but not limited to, p-amino-phenylalanine andreductive amination.

In one embodiment, compositions of relaxin polypeptide that include anunnatural amino acid (such as p-(propargyloxy)-phenyalanine) areprovided. Various compositions comprising p-(propargyloxy)-phenyalanineand, including but not limited to, proteins and/or cells, are alsoprovided. In one aspect, a composition that includes thep-(propargyloxy)-phenyalanine unnatural amino acid, further includes anorthogonal tRNA. The unnatural amino acid can be bonded (including butnot limited to, covalently) to the orthogonal tRNA, including but notlimited to, covalently bonded to the orthogonal tRNA though anamino-acyl bond, covalently bonded to a 3′OH or a 2′OH of a terminalribose sugar of the orthogonal tRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(including but not limited to, amino acids with benzophenone andarylazides (including but not limited to, phenylazide) side chains), forexample, allow for efficient in vivo and in vitro photocrosslinking ofprotein. Examples of photoreactive unnatural amino acids include, butare not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.The protein with the photoreactive unnatural amino acids can then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, including but notlimited to, methyl group, as a probe of local structure and dynamics,including but not limited to, with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2] cycloaddition reaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2nd reactive groupdifferent from the NH2 group normally present in α-amino acids (seeFormula I). A similar non-natural amino acid can be incorporated at thecarboxyl terminus with a 2nd reactive group different from the COOHgroup normally present in α-amino acids (see Formula I).

The unnatural amino acids of the invention may be selected or designedto provide additional characteristics unavailable in the twenty naturalamino acids. For example, unnatural amino acid may be optionallydesigned or selected to modify the biological properties of a protein,e.g., into which they are incorporated. For example, the followingproperties may be optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, solubility, stability,e.g., thermal, hydrolytic, oxidative, resistance to enzymaticdegradation, and the like, facility of purification and processing,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic activity, redox potential,half-life, ability to react with other molecules, e.g., covalently ornoncovalently, and the like.

Structure and Synthesis of Non-Natural Amino Acids: Carbonyl,Carbonyl-Like, Masked Carbonyl, Protected Carbonyl Groups, andHydroxylamine Groups

In some embodiments the present invention provides relaxin linked to awater soluble polymer, e.g., a PEG, by an oxime bond.

Many types of non-naturally encoded amino acids are suitable forformation of oxime bonds. These include, but are not limited to,non-naturally encoded amino acids containing a carbonyl, dicarbonyl, orhydroxylamine group. Such amino acids are described in U.S. PatentPublication Nos. 2006/0194256, 2006/0217532, and 2006/0217289 and WO2006/069246 entitled “Compositions containing, methods involving, anduses of non-natural amino acids and polypeptides,” which areincorporated herein by reference in their entirety. Non-naturallyencoded amino acids are also described in U.S. Pat. Nos. 7,083,970 and7,045,337, which are incorporated by reference herein in their entirety.

Some embodiments of the invention utilize relaxin polypeptides that aresubstituted at one or more positions with a para-acetylphenylalanineamino acid. The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine are described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), incorporated by reference. Othercarbonyl- or dicarbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art. Further, non-limiting exemplarysyntheses of non-natural amino acid that are included herein arepresented in FIGS. 4, 24-34 and 36-39 of U.S. Pat. No. 7,083,970, whichis incorporated by reference herein in its entirety.

Amino acids with an electrophilic reactive group allow for a variety ofreactions to link molecules via nucleophilic addition reactions amongothers. Such electrophilic reactive groups include a carbonyl group(including a keto group and a dicarbonyl group), a carbonyl-like group(which has reactivity similar to a carbonyl group (including a ketogroup and a dicarbonyl group) and is structurally similar to a carbonylgroup), a masked carbonyl group (which can be readily converted into acarbonyl group (including a keto group and a dicarbonyl group)), or aprotected carbonyl group (which has reactivity similar to a carbonylgroup (including a keto group and a dicarbonyl group) upondeprotection). Such amino acids include amino acids having the structureof Formula (IV):

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

J is

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

each R″ is independently H, alkyl, substituted alkyl, or a protectinggroup, or when more than one R″ group is present, two R″ optionally forma heterocycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

each of R₃ and R₄ is independently H, halogen, lower alkyl, orsubstituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally form acycloalkyl or a heterocycloalkyl;

or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkylor heterocycloalkyl comprising at least one carbonyl group, including adicarbonyl group, protected carbonyl group, including a protecteddicarbonyl group, or masked carbonyl group, including a maskeddicarbonyl group:

or the -J-R group together forms a monocyclic or bicyclic cycloalkyl orheterocycloalkyl comprising at least one carbonyl group, including adicarbonyl group, protected carbonyl group, including a protecteddicarbonyl group, or masked carbonyl group, including a maskeddicarbonyl group:

with a proviso that when A is phenylene and each R₃ is H, B is present;and that when A is —(CH₂)₄— and each R₃ is H, B is not —NHC(O)(CH₂CH₂)—;and that when A and B are absent and each R₃ is H, R is not methyl.

In addition, having the structure of Formula (V) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

with a proviso that when A is phenylene, B is present; and that when Ais —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—; and that when A and B areabsent, R is not methyl.

In addition, amino acids having the structure of Formula (VI) areincluded:

wherein:

B is a linker selected from the group consisting of lower alkylene,substituted lower alkylene, lower alkenylene, substituted loweralkenylene, lower heteroalkylene, substituted lower heteroalkylene, —O—,—O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substitutedalkylene)-, —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene orsubstituted alkylene)-, —C(O)—, —C(O)-(alkylene or substitutedalkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)R′, where each R′ is independently H,alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected group, carboxylprotected or a salt thereof. In addition, any of the followingnon-natural amino acids may be incorporated into a non-natural aminoacid polypeptide.

In addition, the following amino acids having the structure of Formula(VII) are included:

wherein

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl; and n is 0 to 8;

with a proviso that when A is —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(VIII) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(IX) are included:

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

wherein each R is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(X) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

each Ra is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)2, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)2, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition to monocarbonyl structures, the non-natural amino acidsdescribed herein may include groups such as dicarbonyl, dicarbonyl like,masked dicarbonyl and protected dicarbonyl groups.

For example, the following amino acids having the structure of Formula(XI) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, O-(alkylene or substitutedalkylene)-, —S—, S-(alkylene or substituted alkylene)-, —S(O)k- where kis 1, 2, or 3, —S(O)k(alkylene or substituted alkylene)-, C(O)—,C(O)-(alkylene or substituted alkylene)-, —C(S)—, C(S)-(alkylene orsubstituted alkylene)-, —N(R′)—, NR′-(alkylene or substitutedalkylene)-, C(O)N(R′)—, CON(R′)-(alkylene or substituted alkylene)-,—CSN(R′)—, CSN(R′)-(alkylene or substituted alkylene)-,N(R′)CO-(alkylene or substituted alkylene)-, N(R′)C(O)O—,S(O)_(k)N(R′)—, N(R′)C(O)N(R′)—, N(R′)C(S)N(R′)—, N(R′)S(O)_(k)N(R′)—,N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, C(R′)2-N═N—, andC(R′)2 N(R′) N(R′)—, where each R′ is independently H, alkyl, orsubstituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(XII) are included:

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)— where kis 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,—C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene orsubstituted alkylene)-, —N(R′)—, —NR′-(alkylene or substitutedalkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-,—CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

wherein each Ra is independently selected from the group consisting ofH, halogen, alkyl, substituted alkyl, —N(R′)2, —C(O)_(k)R′ where k is 1,2, or 3, —C(O)N(R′)2, —OR′, and —S(O)_(k)R′, where each R′ isindependently H, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIII) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIV) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

X1 is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-A) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-B) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

X₁ is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ oneach CR⁸R⁹ group is independently selected from the group consisting ofH, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ cantogether form ═O or a cycloalkyl, or any to adjacent R⁸ groups cantogether form a cycloalkyl.

In addition the following amino acids having the structure of Formula(XV-A) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group isindependently selected from the group consisting of H, alkoxy,alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form ═Oor a cycloalkyl, or any to adjacent R groups can together form acycloalkyl.

In addition, the following amino acids having the structure of Formula(XV-B) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group isindependently selected from the group consisting of H, alkoxy,alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form ═Oor a cycloalkyl, or any to adjacent R groups can together form acycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-A) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-B) are included:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, amino acids having the structure of Formula (XVII) areincluded:

wherein:

A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl;

T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl;

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, amino acids having the structure of Formula (XVIII) areincluded:

wherein:

M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl:

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl;

T₃ is a bond, C(RXR), O, or S, and R is H, halogen, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl:

R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; and

R₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

each R is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl.

In addition, amino acids having the structure of Formula (XIX) areincluded:

wherein:

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl; and

T₃ is O, or S.

In addition, amino acids having the structure of Formula (XX) areincluded:

wherein:

R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl.

In addition, the following amino acids having structures of Formula(XXI) are included:

In some embodiments, a polypeptide comprising a non-natural amino acidis chemically modified to generate a reactive carbonyl or dicarbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et. al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-natural amino acid bearing adjacenthydroxyl and amino groups can be incorporated into the polypeptide as a“masked” aldehyde functionality. For example, 5-hydroxylysine bears ahydroxyl group adjacent to the epsilon amine. Reaction conditions forgenerating the aldehyde typically involve addition of molar excess ofsodium metaperiodate under mild conditions to avoid oxidation at othersites within the polypeptide. The pH of the oxidation reaction istypically about 7.0. A typical reaction involves the addition of about1.5 molar excess of sodium meta periodate to a buffered solution of thepolypeptide, followed by incubation for about 10 minutes in the dark.See, e.g. U.S. Pat. No. 6,423,685.

The carbonyl or dicarbonyl functionality can be reacted selectively witha hydroxylamine-containing reagent under mild conditions in aqueoussolution to form the corresponding oxime linkage that is stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonyl ordicarbonyl group allows for selective modification in the presence ofthe other amino acid side chains. See, e.g., Cornish, V. W., et al., J.Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G.,Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al., Science276:1125-1128 (1997).

Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids

U.S. patent application Ser. No. 11/316,534 (U.S. Publication No.20060189529) is incorporated by reference in its entirety. Thus, thedisclosures provided in Section V (entitled “Non-natural Amino Acids”),Part B (entitled “Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids”), in U.S. patent application Ser.No. 11/316,534 (U.S. Publication No. 20060189529) apply fully to themethods, compositions (including Formulas I-XXXV), techniques andstrategies for making, purifying, characterizing, and using non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides described herein to the same extentas if such disclosures were fully presented herein. U.S. PatentPublication Nos. 2006/0194256, 2006/0217532, and 2006/0217289 and WO2006/069246 entitled “Compositions containing, methods involving, anduses of non-natural amino acids and polypeptides,” are also incorporatedherein by reference in their entirety.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, WI, USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of ordinary skillin the art. For organic synthesis techniques, see, e.g., OrganicChemistry by Fessendon and Fessendon, (1982, Second Edition, WillardGrant Press, Boston Mass.); Advanced Organic Chemistry by March (ThirdEdition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistryby Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press,New York). Additional publications describing the synthesis of unnaturalamino acids include, e.g., WO 2002/085923 entitled “In vivoincorporation of Unnatural Amino Acids;” Matsoukas et al., (1995) J.Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A. (1949) A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. &Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170: Aroulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL- and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, U.S. Patent Publication No. US 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference herein.

A. Carbonyl Reactive Groups

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

Exemplary carbonyl-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group. In some embodiments, n is 1,R₁ is phenyl and R₂ is a simple alkyl (i.e., methyl, ethyl, or propyl)and the ketone moiety is positioned in the para position relative to thealkyl side chain. In some embodiments, n is 1, R1 is phenyl and R₂ is asimple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety ispositioned in the meta position relative to the alkyl side chain.

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art.

In some embodiments, a polypeptide comprising a non-naturally encodedamino acid is chemically modified to generate a reactive carbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992): Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J, and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal. L. K., et al., Science 276:1125-1128(1997).

B. Hydrazine, Hydrazide or Semicarbazide Reactive Groups

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Exemplary hydrazine, hydrazide or semicarbazide-containing amino acidscan be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X, is O, N, or S or not present; R₂ isH, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, n is 4, R₁ is not present, and X is N. In someembodiments, n is 2, R₁ is not present, and X is not present. In someembodiments, n is 1, R₁ is phenyl, X is O, and the oxygen atom ispositioned para to the alphatic group on the aryl ring.

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-L-hydrazideis available from Sigma Chemical (St. Louis, MO). Other amino acids notavailable commercially can be prepared by one of ordinary skill in theart. See, e.g., U.S. Pat. No. 6,281,211, which is incorporated byreference herein.

Polypeptides containing non-naturally encoded amino acids that bearhydrazide, hydrazine or semicarbazide functionalities can be reactedefficiently and selectively with a variety of molecules that containaldehydes or other functional groups with similar chemical reactivity.See, e.g., Shao, J, and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995).The unique reactivity of hydrazide, hydrazine and semicarbazidefunctional groups makes them significantly more reactive towardaldehydes, ketones and other electrophilic groups as compared to thenucleophilic groups present on the 20 common amino acids (including butnot limited to, the hydroxyl group of serine or threonine or the aminogroups of lysine and the N-terminus).

C. Aminooxy-Containing Amino Acids

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J, and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl-containing group suchas a ketone.

Exemplary amino acids containing aminooxy groups can be represented asfollows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10;Y═C(O) or not present; R₂ is H, an amino acid, a polypeptide, or anamino terminus modification group, and R₃ is H, an amino acid, apolypeptide, or a carboxy terminus modification group. In someembodiments, n is 1, R₁ is phenyl, X is O, m is 1, and Y is present. Insome embodiments, n is 2, R₁ and X are not present, m is 0, and Y is notpresent.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G., Life Sci.60: 1635-1641 (1997). Other aminooxy-containing amino acids can beprepared by one of ordinary skill in the art.

D. Azide and Alkyne Reactive Groups

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly alphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occurring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2] cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4. (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containing relaxinpolypeptide can be carried out at room temperature under aqueousconditions by the addition of Cu(II) (including but not limited to, inthe form of a catalytic amount of CuSO₄) in the presence of a reducingagent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe,C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al.,Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agentsinclude, including but not limited to, ascorbate, metallic copper,quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, andan applied electric potential.

In some cases, where a Huisgen [3+2] cycloaddition reaction between anazide and an alkyne is desired, the relaxin polypeptide comprises anon-naturally encoded amino acid comprising an alkyne moiety and thewater soluble polymer to be attached to the amino acid comprises anazide moiety. Alternatively, the converse reaction (i.e., with the azidemoiety on the amino acid and the alkyne moiety present on the watersoluble polymer) can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine).

Exemplary water soluble polymers containing an aryl ester and aphosphine moiety can be represented as follows:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂, R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide. Exemplary water soluble polymers containing athioester and a phosphine moiety can be represented as follows:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

Exemplary alkyne-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10,R₂ is H, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl. X is notpresent, m is 0 and the acetylene moiety is positioned in the paraposition relative to the alkyl side chain. In some embodiments, n is 1,R₁ is phenyl, X is O, m is 1 and the propargyloxy group is positioned inthe para position relative to the alkyl side chain (i.e.,O-propargyl-tyrosine). In some embodiments, n is 1, R₁ and X are notpresent and m is 0 (i.e., proparylglycine).

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,MA). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one of ordinary skill in the art.

Exemplary azide-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, in is 0 and the azide moiety is positioned para to the alkylside chain. In some embodiments, n is 0-4 and R, and X are not present,and m=0. In some embodiments, n is 1, R₁ is phenyl, X is O, m is 2 andthe β-azidoethoxy moiety is positioned in the para position relative tothe alkyl side chain.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, IL). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofordinary skill in the art, including but not limited to, viadisplacement of a suitable leaving group (including but not limited to,halide, mesylate, tosylate) or via opening of a suitably protectedlactone. See, e.g., Advanced Organic Chemistry by March (Third Edition,1985, Wiley and Sons, New York).

E. Aminothiol Reactive Groups

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated into relaxinpolypeptides and then reacted with water soluble polymers comprising analdehyde functionality. In some embodiments, a water soluble polymer,drug conjugate or other payload can be coupled to a relaxin polypeptidecomprising a beta-substituted aminothiol amino acid via formation of thethiazolidine.

F. Additional Reactive Groups

Additional reactive groups and non-naturally encoded amino acids,including but not limited to para-amino-phenylalanine orpara-acetyl-phenylalanine, that can be incorporated into relaxinpolypeptides of the invention are described in the following patentapplications which are all incorporated by reference in their entiretyherein: U.S. Patent Publication No. 2006/0194256, U.S. PatentPublication No. 2006/0217532, U.S. Patent Publication No. 2006/0217289,U.S. Provisional Patent No. 60/755,338; U.S. Provisional Patent No.60/755,711; U.S. Provisional Patent No. 60/755,018; International PatentApplication No. PCT/US06/49397; WO 2006/069246; U.S. Provisional PatentNo. 60/743,041; U.S. Provisional Patent No. 60/743,040; InternationalPatent Application No. PCT/US06/47822; U.S. Provisional Patent No.60/882,819; U.S. Provisional Patent No. 60/882,500; and U.S. ProvisionalPatent No. 60/870,594. These applications also discuss reactive groupsthat may be present on PEG or other polymers, including but not limitedto, hydroxylamine (aminooxy) groups for conjugation.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, includingbut not limited to, for incorporation into a protein. For example, thehigh charge density of α-amino acids suggests that these compounds areunlikely to be cell permeable. Natural amino acids are taken up into theeukaryotic cell via a collection of protein-based transport systems. Arapid screen can be done which assesses which unnatural amino acids, ifany, are taken up by cells. See, e.g., the toxicity assays in. e.g.,U.S. Patent Publication No. US 2004/0198637 entitled “Protein Arrays”which is incorporated by reference herein; and Liu, D. R. & Schultz, P.G. (1999) Progress toward the evolution of an organism with an expandedgenetic code. PNAS United States 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a cell, the invention provides such methods. Forexample, biosynthetic pathways for unnatural amino acids are optionallygenerated in host cell by adding new enzymes or modifying existing hostcell pathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, for example, in Genbank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the WorldWide Web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, those identified through functionalgenomics, and molecular evolution and design. Diversa Corporation(available on the World Wide Web at diversa.com) also providestechnology for rapidly screening libraries of genes and gene pathways,including but not limited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution,modulating immunogenicity, modulating protease resistance, etc. Proteinsthat include an unnatural amino acid can have enhanced or even entirelynew catalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient(including but not limited to, a pharmaceutically acceptable excipient)can also be present with the protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In one aspect, the post-translational modification includesattachment of an oligosaccharide (including but not limited to,(GlcNAc-Man)2-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table A which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.

TABLE A Examples of oligosaccharides through GLCNAC-linkage Type BaseStructure HIGH- MANNOSE

HYBRID

COMPLEX

XYLOSE

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,prorelaxin or a variant or analog thereof), assembly into a multisubunitprotein or macromolecular assembly, translation to another site in thecell (including but not limited to, to organelles, such as theendoplasmic reticulum, the Golgi apparatus, the nucleus, lysosomes,peroxisomes, mitochondria, chloroplasts, vacuoles, etc., or through thesecretory pathway). In certain embodiments, the protein comprises asecretion or localization sequence, an epitope tag, a FLAG tag, apolyhistidine tag, a GST fusion, or the like.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7, all of which are incorporated byreference herein. This allows the selective labeling of virtually anyprotein with a host of reagents including fluorophores, crosslinkingagents, saccharide derivatives and cytotoxic molecules. Sec also, U.S.Pat. No. 6,927,042 entitled “Glycoprotein synthesis,” which isincorporated by reference herein. Post-translational modifications,including but not limited to, through an azido amino acid, can also madethrough the Staudinger ligation (including but not limited to, withtriarylphosphine reagents). See, e.g., Kiick et al., (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, including but not limited to,containing an azide or alkynyl moiety into proteins in response to aselector codon. These amino acid side chains can then be modified by,including but not limited to, a Huisgen [3+2] cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa. A., Wiley. NewYork, p. 1-176) with, including but not limited to, alkynyl or azidederivatives, respectively. Because this method involves a cycloadditionrather than a nucleophilic substitution, proteins can be modified withextremely high selectivity. This reaction can be carried out at roomtemperature in aqueous conditions with excellent regioselectivity(1,4>1,5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tornoe, et al., (2002) J. Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. Another method that can be used is the ligand exchange ona bisarsenic compound with a tetracysteine motif, see, e.g., Griffin, etal., (1998) Science 281:269-272.

A molecule that can be added to a protein of the invention through a[3+2] cycloaddition includes virtually any molecule with an azide oralkynyl derivative. Molecules include, but are not limited to, dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(including but not limited to, derivatives of polyethylene glycol),photocrosslinkers, cytotoxic compounds, affinity labels, derivatives ofbiotin, resins, beads, a second protein or polypeptide (or more),polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. Thesemolecules can be added to an unnatural amino acid with an alkynyl group,including but not limited to, p-propargyloxyphenylalanine, or azidogroup, including but not limited to, p-azido-phenylalanine,respectively.

In Vivo Generation of Relaxin Polypeptides ComprisingNon-Naturally-Encoded Amino Acids

The relaxin polypeptides of the invention can be generated in vivo usingmodified tRNA and tRNA synthetases to add to or substitute amino acidsthat are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Pat. Nos. 7,045,337 and 7,083,970 which are incorporated byreference herein. These methods involve generating a translationalmachinery that functions independently of the synthetases and tRNAsendogenous to the translation system (and are therefore sometimesreferred to as “orthogonal”). Typically, the translation systemcomprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNAsynthetase (O—RS). Typically, the O—RS preferentially aminoacylates theO-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO—RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Pat. Nos. 7,045,337 and7,083,970, each incorporated herein by reference. Corresponding O-tRNAmolecules for use with the O—RSs are also described in U.S. Pat. Nos.7,045,337 and 7,083,970 which are incorporated by reference herein.Additional examples of O-tRNA/aminoacyl-tRNA synthetase pairs aredescribed in WO 2005/007870, WO 2005/007624; and WO 2005/019415.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027(2002). Exemplary O—RS sequences for p-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. Pat. No.7,083,970 which is incorporated by reference herein. Exemplary O-tRNAsequences suitable for use in the present invention include, but are notlimited to, nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S.Pat. No. 7,083,970, which is incorporated by reference herein. Otherexamples of O-tRNA/aminoacyl-tRNA synthetase pairs specific toparticular non-naturally encoded amino acids are described in U.S. Pat.No. 7,045,337 which is incorporated by reference herein. O—RS and O-tRNAthat incorporate both keto- and azide-containing amino acids in S.cerevisiae are described in Chin, J. W., et al., Science 301:964-967(2003).

Several other orthogonal pairs have been reported. Glutaminyl (see,e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci. U.S.A96:47804785), aspartyl (see, e.g., Pastrnak, M., et al., (2000) Helv.Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., et al.,(1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K., etal., (2001) Proc. Natl. Acad. Sci. U.S.A 98:2268-2273) systems derivedfrom S. cerevisiae tRNA's and synthetases have been described for thepotential incorporation of unnatural amino acids in E. coli. Systemsderived from the E. coli glutaminyl (see, e.g., Kowal, A. K., et al.,(2001) Proc. Natl. Acad. Sci. U.S.A 98:2268-2273) and tyrosyl (see,e.g., Edwards. H., and Schimmel, P. (1990) Mol. Cell. Biol.10:1633-1641) synthetases have been described for use in S. cerevisiae.The E. coli tyrosyl system has been used for the incorporation of3-iodo-L-tyrosine in vivo, in mammalian cells. See, Sakamoto, K., etal., (2002) Nucleic Acids Res. 30:4692-4699.

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the relaxin polynucleotide coding sequence using mutagenesis methodsknown in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

Methods for generating components of the protein biosynthetic machinery,such as O—RSs, O-tRNAs, and orthogonal O-tRNA/O—RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003). Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. Pat. No.7,045,337, which is incorporated by reference herein. Methods forselecting an orthogonal tRNA-tRNA synthetase pair for use in in vivotranslation system of an organism are also described in U.S. Pat. Nos.7,045,337 and 7,083,970 which are incorporated by reference herein. PCTPublication No. W0 04/035743 entitled “Site Specific Incorporation ofKeto Amino Acids into Proteins,” which is incorporated by referenceherein in its entirety, describes orthogonal RS and tRNA pairs for theincorporation of keto amino acids. PCT Publication No. WO 04/094593entitled “Expanding the Eukaryotic Genetic Code.” which is incorporatedby reference herein in its entirety, describes orthogonal RS and tRNApairs for the incorporation of non-naturally encoded amino acids ineukaryotic host cells.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O—RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus. P.furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like, or aeukaryotic organism; (b) selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that aminoacylate an orthogonal tRNA(O-tRNA) in the presence of a non-naturally encoded amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-naturally encoded amino acid, thereby providing the at least onerecombinant O—RS; wherein the at least one recombinant O—RSpreferentially aminoacylates the O-tRNA with the non-naturally encodedamino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, including but notlimited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. For example, the mutant RSscan be generated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, rational designand by other methods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, including but notlimited to, that aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-naturally encoded amino acid and a natural amino acid,includes: introducing a positive selection or screening marker,including but not limited to, an antibiotic resistance gene, or thelike, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, including but not limited to, an amber,ochre, or opal codon; growing the plurality of cells in the presence ofa selection agent; identifying cells that survive (or show a specificresponse) in the presence of the selection and/or screening agent bysuppressing the at least one selector codon in the positive selection orscreening marker, thereby providing a subset of positively selectedcells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the non-naturally encoded amino acid includes:introducing a negative selection or screening marker with the pool ofactive (optionally mutant) RSs from the positive selection or screeninginto a plurality of cells of a second organism, wherein the negativeselection or screening marker comprises at least one selector codon(including but not limited to, an antibiotic resistance gene, includingbut not limited to, a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a first medium supplemented with the non-naturally encodedamino acid and a screening or selection agent, but fail to survive or toshow the specific response in a second medium not supplemented with thenon-naturally encoded amino acid and the selection or screening agent,thereby providing surviving cells or screened cells with the at leastone recombinant O—RS. For example, a CAT identification protocoloptionally acts as a positive selection and/or a negative screening indetermination of appropriate O—RS recombinants. For instance, a pool ofclones is optionally replicated on growth plates containing CAT (whichcomprises at least one selector codon) either with or without one ormore non-naturally encoded amino acid. Colonies growing exclusively onthe plates containing non-naturally encoded amino acids are thusregarded as containing recombinant O—RS. In one aspect, theconcentration of the selection (and/or screening) agent is varied. Insome aspects the first and second organisms are different. Thus, thefirst and/or second organism optionally comprises: a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacterium, a eubacterium, a plant, an insect, a protist, etc. Inother embodiments, the screening marker comprises a fluorescent orluminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RSsincludes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection or screeningmarker, wherein the negative selection or screening marker comprises atleast one selector codon (including but not limited to, a toxic markergene, including but not limited to, a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a first medium not supplemented with the non-naturally encoded aminoacid, but fail to survive or show a specific screening response in asecond medium supplemented with the non-naturally encoded amino acid,thereby providing surviving or screened cells with the at least onerecombinant O—RS, wherein the at least one recombinant O—RS is specificfor the non-naturally encoded amino acid. In one aspect, the at leastone selector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O—RS) can further comprise: (d)isolating the at least one recombinant O—RS; (e) generating a second setof O—RS (optionally mutated) derived from the at least one recombinantO—RS; and, (f) repeating steps (b) and (c) until a mutated O—RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, including but notlimited to, at least about two times. In one aspect, the second set ofmutated O—RS derived from at least one recombinant O—RS can be generatedby mutagenesis, including but not limited to, random mutagenesis,site-specific mutagenesis, recombination or a combination thereof.

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-naturally encoded amino acid or an analogue. In one embodiment, themutated synthetase is displayed on a cell surface, on a phage display orthe like.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,including but not limited to, a suppressor tRNA, from a first organism;(b) selecting (including but not limited to, negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase (RS) from a secondorganism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS (O—RS), thereby providingat least one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O—RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-naturally encoded amino acid, whereinthe non-naturally encoded amino acid is biosynthesized in vivo eithernaturally or through genetic manipulation. The non-naturally encodedamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O—RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, □-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O—RS, and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O—RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O—RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O—RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant O-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the O—RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anon-naturally encoded amino acid and a natural amino acid, therebyproviding a pool of active (optionally mutant) RSs; and, (f) negativelyselecting or screening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the non-naturally encoded amino acid, thereby providing theat least one specific O-tRNA/O—RS pair, wherein the at least onespecific O-tRNA/O—RS pair comprises at least one recombinant O—RS thatis specific for the non-naturally encoded amino acid and the at leastone recombinant O-tRNA. Specific O-tRNA/O—RS pairs produced by themethods are included. For example, the specific O-tRNA/O—RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism:introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, orthe like. Alternatively, the organisms optionally comprise a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like. In another embodiment, the second organism is a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, including but not limited to, a yeast, a animalcell, a plant cell, a fungus, a mammalian cell, or the like. In variousembodiments the first and second organisms are different.

Location of Non-Naturally-Occurring Amino Acids in Relaxin Polypeptides

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into relaxin polypeptides. One ormore non-naturally-occurring amino acids may be incorporated at aparticular position which does not disrupt activity of the polypeptide.This can be achieved by making “conservative” substitutions, includingbut not limited to, substituting hydrophobic amino acids withhydrophobic amino acids, bulky amino acids for bulky amino acids,hydrophilic amino acids for hydrophilic amino acids and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the relaxin polypeptide. It is readily apparent tothose of ordinary skill in the art that any position of the polypeptidechain is suitable for selection to incorporate a non-naturally encodedamino acid, and selection may be based on rational design or by randomselection for any or no particular desired purpose. Selection of desiredsites may be for producing a relaxin molecule having any desiredproperty or activity, including but not limited to, agonists,super-agonists, inverse agonists, antagonists, receptor bindingmodulators, receptor activity modulators, dimer or multimer formation,no change to activity or property compared to the native molecule, ormanipulating any physical or chemical property of the polypeptide suchas solubility, aggregation, or stability. For example, locations in thepolypeptide required for biological activity of relaxin polypeptides canbe identified using point mutation analysis, alanine scanning,saturation mutagenesis and screening for biological activity, or homologscanning methods known in the art. Other methods can be used to identifyresidues for modification of relaxin polypeptides include, but are notlimited to, sequence profiling (Bowie and Eisenberg, Science 253(5016):164-70. (1991)), rotamer library selections (Dahiyat and Mayo, ProteinSci 5(5): 895-903 (1996); Dahiyat and Mayo, Science 278(5335): 82-7(1997); Desjarlais and Handel, Protein Science 4: 2006-2018 (1995);Harbury et al, PNAS USA 92(18): 8408-8412 (1995); Kono et al., Proteins:Structure, Function and Genetics 19: 244-255 (1994); Hellinga andRichards, PNAS USA 91: 5803-5807 (1994)); and residue pair potentials(Jones, Protein Science 3: 567-574, (1994)), and rational design usingProtein Design Automation® technology. (See U.S. Pat. Nos. 6,188,965;6,269,312; 6,403,312; WO98/47089, which are incorporated by reference).Residues that are critical for relaxin bioactivity, residues that areinvolved with pharmaceutical stability, antibody epitopes, or receptorbinding residues may be mutated. U.S. Pat. Nos. 5,580,723; 5,834,250;6,013,478; 6,428,954; and 6,451,561, which are incorporated by referenceherein, describe methods for the systematic analysis of the structureand function of polypeptides such as relaxin by identifying activedomains which influence the activity of the polypeptide with a targetsubstance. Residues other than those identified as critical tobiological activity by alanine or homolog scanning mutagenesis may begood candidates for substitution with a non-naturally encoded amino aciddepending on the desired activity sought for the polypeptide.Alternatively, the sites identified as critical to biological activitymay also be good candidates for substitution with anon-naturally encodedamino acid, again depending on the desired activity sought for the polypeptide. Another alternative would be to simply make serialsubstitutions in each position on the polypeptide chain with anon-naturally encoded amino acid and observe the effect on theactivities of the polypeptide. It is readily apparent to those ofordinary skill in the art that any means, technique, or method forselecting a position for substitution with a non-natural amino acid intoany polypeptide is suitable for use in the present invention.

The structure and activity of mutants of relaxin polypeptides thatcontain deletions can also be examined to determine regions of theprotein that are likely to be tolerant of substitution withanon-naturally encoded amino acid. In a similar manner, proteasedigestion and monoclonal antibodies can be used to identify regions ofrelaxin that are responsible for binding the relaxin receptor. Onceresidues that are likely to be intolerant to substitution withnon-naturally encoded amino acids have been eliminated, the impact ofproposed substitutions at each of the remaining positions can beexamined. Models may be generated from the three-dimensional crystalstructures of other relaxin family members and relaxin receptors.Protein Data Bank (PDB, available on the World Wide Web at rcsb.org) isa centralized database containing three-dimensional structural data oflarge molecules of proteins and nucleic acids. Models may be madeinvestigating the secondary and tertiary structure of polypeptides, ifthree-dimensional structural data is not available. Thus, those ofordinary skill in the art can readily identify amino acid positions thatcan be substituted with non-naturally encoded amino acids.

Relaxin A Chain complex solvent accessibility chain A/B interfaceResidue Residue Mainchain Sidechain Residue Mainchain Sidechain NameResidue # Average Average Average Average Average Average LEU 2 2.3939272.336841 2.451013 0.00913 0.018259 0 TYR 3 1.05745 1.412109 0.8801210.448218 0.164923 0.589865 SER 4 2.039557 1.726208 2.666253 0.0947010.127334 0.029435 ALA 5 1.47603 1.32887 2.064669 0.040922 0.051152 0 LEU6 0.520247 0.621696 0.418798 0.174242 0.189235 0.159248 ALA 7 0.4760430.450361 0.578769 0.512186 0.474717 0.662064 ASN 8 1.252271 0.8659121.638629 0.32958 0.460124 0.199036 LYS 9 0.886439 0.727133 1.0138840.424786 0.531522 0.339397 CYS 10 0.307912 0.414588 0.094561 0.7313480.802621 0.588801 CYS 11 0.791671 0.774527 0.825959 1.267628 1.1703851.462113 HIS 12 1.644891 1.389182 1.815363 0.902367 0.910146 0.897182VAL 13 1.432029 1.329367 1.568911 0.865782 1.041144 0.631966 GLY 140.721258 0.721258 0 0.814179 0.814179 0 CYS 15 0.405285 0.4708220.274212 0.79239 0.856379 0.664412 THR 16 0.511268 0.367613 0.7028081.096358 1.011865 1.209014 LYS 17 0.376592 0.233542 0.491032 2.6917421.414171 3.713799 ARG 18 0.97893 0.478638 1.264811 1.626733 1.129031.911135 SER 19 0.416177 0.424198 0.405482 0.476934 0.49423 0.453873 LEU20 0.155542 0.267306 0.043778 0.735924 0.711222 0.760627 ALA 21 0.4786450.507787 0.362075 1.105314 1.045545 1.344386 ARG 22 1.273184 1.1021021.370945 0.551495 0.572567 0.539454 PHE 23 1.054989 1.153619 0.9986290.428106 0.829454 0.198764 CYS 24 1.352353 1.506838 1.146374 2.074392.22276 1.876563 Relaxin B Chain complex solvent accessibility chain A/Binterface Residue Residue Mainchain Sidechain Residue MainchainSidechain Name Residue # Average Average Average Average Average AverageSER 2 2.241497 2.362674 1.999142 1.249697 1.108926 1.531239 TRP 31.036157 1.320435 0.922445 0.774516 1.009561 0.680498 MET 4 0.7659030.783709 0.748097 1.378683 1.318601 1.438766 GLU 5 1.401968 1.1021951.641786 1.60857 1.38927 1.784009 GLU 6 1.209446 0.935167 1.4288690.994341 1.251666 0.788481 VAL 7 1.123345 1.08403 1.175766 1.9174981.613757 2.322484 ILE 8 0.478044 0.594216 0.361873 1.329975 1.7262640.933686 LYS 9 1.135226 0.723724 1.464427 2.332893 1.427852 3.056925 LEU10 0.579 0.504927 0.653072 0.906043 1.195675 0.616411 CYS 11 0.8628620.806072 0.976442 1.030881 0.795964 1.500714 GLY 12 1.089858 1.089858 00.604306 0.604306 0 ARG 13 3.079311 1.482092 3.992007 0.024047 0.0661280 GLU 14 1.46883 0.992251 1.680643 0.137036 0.204496 0.107054 LEU 150.378917 0.508368 0.249467 0.743117 0.470105 1.016129 VAL 16 1.0180060.820163 1.281796 0.279417 0.29034 0.264852 ARG 17 1.660023 0.9379772.072621 0.048147 0.132403 0 ALA 18 0.51055 0.547669 0.362075 0.3790880.387335 0.346101 GLN 19 0.436502 0.481692 0.400351 1.12185 0.7338731.432231 ILE 20 1.321723 0.993689 1.649758 0.294635 0.473331 0.115939ALA 21 1.001017 0.958569 1.170807 0.362917 0.40431 0.197346 ILE 220.381207 0.565133 0.197281 0.689578 0.665212 0.713943 CYS 23 0.8883020.931778 0.80135 0.782559 0.685197 0.977284 GLY 24 1.608804 1.608804 00.25224 0.25224 0 MET 25 1.419412 1.489301 1.349524 0.239722 0.185720.293724 SER 26 1.07028 1.249672 0.711497 0.582382 0.433677 0.879791 THR27 2.199516 2.265998 2.110873 0.228179 0.155968 0.32446 TRP 28 4.741673.651826 4.971111 0 0 0

In some embodiments, the relaxin polypeptides of the invention compriseone or more non-naturally encoded amino acids positioned in a region ofthe protein that does not disrupt the structure of the polypeptide.

Exemplary, residues of incorporation of a non-naturally encoded aminoacid may be those that are excluded from potential receptor bindingregions, may be fully or partially solvent exposed, have minimal or nohydrogen-bonding interactions with nearby residues, may be minimallyexposed to nearby reactive residues, may be on one or more of theexposed faces, may be a site or sites that are juxtaposed to a secondrelaxin, or other molecule or fragment thereof, may be in regions thatare highly flexible, or structurally rigid, as predicted by thethree-dimensional, secondary, tertiary, or quaternary structure ofrelaxin, bound or unbound to its receptor, or coupled or not coupled toanother biologically active molecule, or may modulate the conformationof the relaxin itself or a dimer or multimer comprising one or morerelaxin, by altering the flexibility or rigidity of the completestructure as desired.

One of ordinary skill in the art recognizes that such analysis ofrelaxin enables the determination of which amino acid residues aresurface exposed compared to amino acid residues that are buried withinthe tertiary structure of the protein. Therefore, it is an embodiment ofthe present invention to substitute a non-naturally encoded amino acidfor an amino acid that is a surface exposed residue.

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in relaxin: inthe A chain before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 (i.e., atthe carboxyl terminus of the protein), and any combination thereof (SEQID NO: 4 or the corresponding position in SEQ ID NO: 1, 2, 3, or 15) orin the B chain before position 1 (i.e. at the N-terminus), 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 5 or 6 or the correspondingposition in SEQ ID NO: 1, 2, 3, or 15).

An examination of the crystal structure of relaxin and its interactionwith the relaxin receptor can indicate which certain amino acid residueshave side chains that are fully or partially accessible to solvent. Theside chain of a non-naturally encoded amino acid at these positions maypoint away from the protein surface and out into the solvent.

In some embodiments, the non-naturally encoded amino acid at one or moreof these positions is linked to a water soluble polymer, including butnot limited to, positions: in the A chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22 (i.e., at the carboxyl terminus of the protein), andany combination thereof (SEQ ID NO: 4 or the corresponding position inSEQ ID NO: 1, 2, 3, or 15) or in the B chain before position 1 (i.e. atthe N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 5or 6 or the corresponding position in SEQ ID NO: 1, 2, 3, or 15).

A wide variety of non-naturally encoded amino acids can be substitutedfor, or incorporated into, a given position in a relaxin polypeptide. Ingeneral, a particular non-naturally encoded amino acid is selected forincorporation based on an examination of the three dimensional crystalstructure of a relaxin polypeptide or other relaxin family member orrelaxin analog with its receptor, a preference for conservativesubstitutions (i.e., aryl-based non-naturally encoded amino acids, suchas p-acetylphenylalanine or O-propargyltyrosine substituting for Phe,Tyr or Trp), and the specific conjugation chemistry that one desires tointroduce into the relaxin polypeptide (e.g., the introduction of4-azidophenylalanine if one wants to effect a Huisgen [3+2]cycloaddition with a water soluble polymer bearing an alkyne moiety or aamide bond formation with a water soluble polymer that bears an arylester that, in turn, incorporates a phosphine moiety)

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove, or any other desirable compound or substance) that comprises asecond reactive group. The first reactive group reacts with the secondreactive group to attach the molecule to the unnatural amino acidthrough a [3+2] cycloaddition. In one embodiment, the first reactivegroup is an alkynyl or azido moiety and the second reactive group is anazido or alkynyl moiety. For example, the first reactive group is thealkynyl moiety (including but not limited to, in unnatural amino acidp-propargyloxyphenylalanine) and the second reactive group is the azidomoiety. In another example, the first reactive group is the azido moiety(including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within therelaxin polypeptide to affect other biological traits of the relaxinpolypeptide. In some cases, the other additions, substitutions ordeletions may increase the stability (including but not limited to,resistance to proteolytic degradation) of the relaxin polypeptide orincrease affinity of the relaxin polypeptide for its receptor. In somecases, the other additions, substitutions or deletions may increase thepharmaceutical stability of the relaxin polypeptide. In some cases, theother additions, substitutions or deletions may enhance the anti-viralactivity of the relaxin polypeptide. In some cases, the other additions,substitutions or deletions may increase the solubility (including butnot limited to, when expressed in E. coli or other host cells) of therelaxin polypeptide. In some embodiments additions, substitutions ordeletions may increase the relaxin polypeptide solubility followingexpression in E. coli or other recombinant host cells. In someembodiments sites are selected for substitution with a naturally encodedor non-natural amino acid in addition to another site for incorporationof a non-natural amino acid that results in increasing the polypeptidesolubility following expression in E. coli or other recombinant hostcells. In some embodiments, the relaxin polypeptides comprise anotheraddition, substitution or deletion that modulates affinity for therelaxin polypeptide receptor, binding proteins, or associated ligand,modulates signal transduction after binding to the relaxin receptor,modulates circulating half-life, modulates release or bio-availability,facilitates purification, or improves or alters a particular route ofadministration. In some embodiments, the relaxin polypeptides comprisean addition, substitution or deletion that increases the affinity of therelaxin variant for its receptor. Similarly, relaxin polypeptides cancomprise chemical or enzyme cleavage sequences, protease cleavagesequences, reactive groups, antibody-binding domains (including but notlimited to, FLAG or poly-His) or other affinity based sequences(including, but not limited to, FLAG, poly-His, GST, etc.) or linkedmolecules (including, but not limited to, biotin) that improve detection(including, but not limited to, GFP), purification, transport throughtissues or cell membranes, prodrug release or activation, relaxin sizereduction, or other traits of the polypeptide.

In some embodiments, the substitution of a non-naturally encoded aminoacid generates a relaxin antagonist. In some embodiments, anon-naturally encoded amino acid is substituted or added in a regioninvolved with receptor binding. In some embodiments, relaxin antagonistscomprise at least one substitution that cause relaxin to act as anantagonist. In some embodiments, the relaxin antagonist comprises anon-naturally encoded amino acid linked to a water soluble polymer thatis present in a receptor binding region of the relaxin molecule.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids aresubstituted with one or more non-naturally-encoded amino acids. In somecases, the relaxin polypeptide further includes 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more substitutions of one or more non-naturally encoded aminoacids for naturally-occurring amino acids. For example, in someembodiments, one or more residues in relaxin are substituted with one ormore non-naturally encoded amino acids. In some cases, the one or morenon-naturally encoded residues are linked to one or more lower molecularweight linear or branched PEGs, thereby enhancing binding affinity andcomparable serum half-life relative to the species attached to a single,higher molecular weight PEG.

In some embodiments, up to two of the following residues of relaxin aresubstituted with one or more non-naturally-encoded amino acids.

Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned relaxin polynucleotide, onetypically subclones polynucleotides encoding a relaxin polypeptide ofthe invention into an expression vector that contains a strong promoterto direct transcription, a transcription/translation terminator, and iffor a nucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are known tothose of ordinary skill in the art and described, e.g., in Sambrook etal. and Ausubel et al.

Bacterial expression systems for expressing relaxin polypeptides of theinvention are available in, including but not limited to, E. coli,Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235(1983): Mosbach et al., Nature 302:543-545 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are known to thoseof ordinary skill in the art and are also commercially available. Incases where orthogonal tRNAs and aminoacyl tRNA synthetases (describedabove) are used to express the relaxin polypeptides of the invention,host cells for expression are selected based on their ability to use theorthogonal components. Exemplary host cells include Gram-positivebacteria (including but not limited to B. brevis. B. subtilis, orStreptomyces) and Gram-negative bacteria (E. coli, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida), as well asyeast and other eukaryotic cells. Cells comprising O-tRNA/O—RS pairs canbe used as described herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 nl to about 100 L or more). Theproduction of large quantities (including but not limited to, greaterthat that typically possible with other methods, including but notlimited to, in vitro translation) of a protein in a eukaryotic cellincluding at least one unnatural amino acid is a feature of theinvention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

A number of vectors suitable for expression of relaxin are commerciallyavailable. Useful expression vectors for eukaryotic hosts, include butare not limited to, vectors comprising expression control sequences fromSV40, bovine papilloma virus, adenovirus and cytomegalovirus. Suchvectors include pCDNA3.1(+)\Hyg (Invitrogen, Carlsbad. Calif., USA) andpCI-neo (Stratagene, La Jolla, Calif., USA). Bacterial plasmids, such asplasmids from E. coli, including pBR322, pET3a and pET12a, wider hostrange plasmids, such as RP4, phage DNAs, e.g., the numerous derivativesof phage lambda, e.g., NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages may be used. The 2p plasmid andderivatives thereof, the POT1 vector (U.S. Pat. No. 4,931,373 which isincorporated by reference), the pJSO37 vector described in (Okkels, Ann.New York Aced. Sci. 782, 202 207, 1996) and pPICZ A, B or C (Invitrogen)may be used with yeast host cells. For insect cells, the vectors includebut are not limited to, pVL941, pBG311 (Cate et al., “Isolation of theBovine and Human Genes for Mullerian Inhibiting Substance and Expressionof the Human Gene In Animal Cells”, Cell, 45, pp. 685 98 (1986),pBluebac 4.5 and pMelbac (Invitrogen, Carlsbad, CA).

The nucleotide sequence encoding a relaxin polypeptide may or may notalso include sequence that encodes a signal peptide. The signal peptideis present when the polypeptide is to be secreted from the cells inwhich it is expressed. Such signal peptide may be any sequence. Thesignal peptide may be prokaryotic or eukaryotic. Coloma. M (1992) J.Imm. Methods 152:89 104) describe a signal peptide for use in mammaliancells (murine Ig kappa light chain signal peptide). Other signalpeptides include but are not limited to, the α-factor signal peptidefrom S. cerevisiae (U.S. Pat. No. 4,870,008 which is incorporated byreference herein), the signal peptide of mouse salivary amylase (O.Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modifiedcarboxypeptidase signal peptide (L. A. Valls et al., Cell 48, 1987, pp.887-897), the yeast BARI signal peptide (WO 87/02670, which isincorporated by reference herein), and the yeast aspartic protease 3(YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp.127-137). A relaxin polypeptide sequence of the disclosure or encodingpolynucleotide may include a natural relaxin signal peptide or codingsequence, such as MPRLFFFHLLGVCLLLNQFSRAVA (SwissProt accession noP04090) or any natural sequence variant thereof e.g., as describedabove.

Examples of suitable mammalian host cells are known to those of ordinaryskill in the art. Such host cells may be Chinese hamster ovary (CHO)cells, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cells (COS) (e.g. COS 1(ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), BabyHamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), andhuman cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells intissue culture. These cell lines and others are available from publicdepositories such as the American Type Culture Collection, Rockville.Md. In order to provide improved glycosylation of the relaxinpolypeptide, a mammalian host cell may be modified to expresssialyltransferase, e.g. 1,6-sialyltransferase, e.g. as described in U.S.Pat. No. 5,047,335, which is incorporated by reference herein.

Methods for the introduction of exogenous DNA into mammalian host cellsinclude but are not limited to, calcium phosphare-mediated transfection,electroporation, DEAE-dextran mediated transfection, liposome-mediatedtransfection, viral vectors and the transfection methods described byLife Technologies Ltd, Paisley, UK using Lipofectamin 2000 and RocheDiagnostics Corporation, Indianapolis, USA using FuGENE 6. These methodsare well known in the art and are described by Ausbel et al. (eds.),1996, Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, USA. The cultivation of mammalian cells may be performed accordingto established methods, e.g. as disclosed in (Animal Cell Biotechnology,Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc.Totowa, N.J., USA and Harrison Mass. and Rae IF, General Techniques ofCell Culture, Cambridge University Press 1997).

Expression Systems, Culture, and Isolation

Relaxin polypeptides may be expressed in any number of suitableexpression systems including, for example, yeast, insect cells,mammalian cells, and bacteria. A description of exemplary expressionsystems is provided below.

Yeast

As used herein, the term “yeast” includes any of the various yeastscapable of expressing a gene encoding a relaxin polypeptide. Such yeastsinclude, but are not limited to, ascosporogenous yeasts (Endomycetales),basidiosporogenous yeasts and yeasts belonging to the Fungi imperfecti(Blastomycetes) group. The ascosporogenous yeasts are divided into twofamilies, Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies. Schizosaccharomycoideae (e.g., genusSchizosaccharomyces). Nadsonioideae, Lipomycoideae and Saccharomycoideae(e.g., genera Pichia, Kluyveromyces and Saccharomyces). Thebasidiosporogenous yeasts include the genera Leucosporidium,Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeastsbelonging to the Fungi Imperfecti (Blastomycetes) group are divided intotwo families, Sporobolomycetaceae (e.g., genera Sporobolomyces andBullera) and Cryptococcaceae (e.g., genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula. Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis,S. oviformis, K. lactis, K. fragilis, C. albicans, C. maltosa, and H.polymorpha.

The selection of suitable yeast for expression of relaxin polypeptidesis within the skill of one of ordinary skill in the art. In selectingyeast hosts for expression, suitable hosts may include those shown tohave, for example, good secretion capacity, low proteolytic activity,good secretion capacity, good soluble protein production, and overallrobustness. Yeast are generally available from a variety of sourcesincluding, but not limited to, the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, CA), and the American Type Culture Collection (“ATCC”)(Manassas, VA).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a relaxin polypeptide, areincluded in the progeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302);K. fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIOTECHNOLOGY (NY) (1990) 8:135); P. guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376):Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and Y.lipolytica; A. nidulans (Ballance et al., BIOCHEM. BIOPHYS. RES. COMMUN.(1983) 112:284-89; Tilburn et al., GENE (1983) 26:205-221; and Yelton etal., PROC. NATL. ACAD. SCI. USA (1984) 81:1470-74); A. niger (Kelly andHynes, EMBO J. (1985) 4:475-479); T. reesia (EP 0 244 234); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357), each incorporated by reference herein.

Control sequences for yeast vectors are known to those of ordinary skillin the art and include, but are not limited to, promoter regions fromgenes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH): hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:12073); and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1969) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein. Other examples ofhybrid promoters include promoters that consist of the regulatorysequences of the ADH2, GAL4, GAL10, or PHO5 genes, combined with thetranscriptional activation region of a glycolytic enzyme gene such asGAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2μ plasmid origin is suitable for yeast.A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly. Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are known to thoseof ordinary skill in the art, and typically include, but are not limitedto, either the transformation of spheroplasts or of intact yeast hostcells treated with alkali cations. For example, transformation of yeastcan be carried out according to the method described in Hsiao et al.,PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al., J.BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare known to those of ordinary skill in the art. See generally U.S.Patent Publication No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Pat. Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/07862; WO 98/37208; and WO 98/26080;European Patent Applications EP 0 946 736; EP 0 732 403; EP 0 480 480;WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164 556.See also Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992) 62(1-2):79-93;Romanos et al., YEAST (1992) 8(6):423-488; Goeddel, METHODS INENZYMOLOGY (1990) 185:3-7, each incorporated by reference herein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods knownto those of ordinary skill in the art. The fermentation methods may beadapted to account for differences in a particular yeast host's carbonutilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113,incorporated by reference herein.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein.

Baculovirus-Infected Insect Cells

The term “insect host” or “insect host cell” refers to a insect that canbe, or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original insect hostcell that has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellthat are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a relaxin polypeptide, are included in the progeny intended bythis definition. Baculovirus expression of relaxin polypeptides isuseful in the present invention and the use of rDNA technology,polypeptides or precursors thereof because relaxin may be biosynthesizedin any number of host cells including bacteria, mammalian cells, insectcells, yeast or fungi. An embodiment of the present invention includesbiosynthesis of relaxin, modified relaxin, relaxin polypeptides, orrelaxin analogs in bacteria, yeast or mammalian cells. Anotherembodiment of the present invention involves biosynthesis done in E.coli or a yeast. Examples of biosynthesis in mammalian cells andtransgenic animals are described in Hakola, K. [Molecular and CellularEndocrinology, 127:59-69, (1997)].

The selection of suitable insect cells for expression of relaxinpolypeptides is known to those of ordinary skill in the art. Severalinsect species are well described in the art and are commerciallyavailable including Aedes aegypti, Bombyx mori, Drosophila melanogaster,Spodoptera frugiperda, and Trichoplusia ni. In selecting insect hostsfor expression, suitable hosts may include those shown to have, interalia, good secretion capacity, low proteolytic activity, and overallrobustness. Insect are generally available from a variety of sourcesincluding, but not limited to, the Insect Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, CA); and the American Type Culture Collection (“ATCC”)(Manassas, VA).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample. Invitrogen Corp. (Carlsbad, CA). These techniques are generallyknown to those of ordinary skill in the art and fully described inSUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO.1555 (1987), herein incorporated by reference. See also, RICHARDSON, 39METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSION PROTOCOLS (1995);AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 16.9-16.11(1994): KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORY GUIDE(1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is known to those of ordinaryskill in the art. See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216;6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932;6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676;5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220;5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO89/01037; WO 88/07082, which are incorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographacalifornicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, O'Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, VIROLOGY 170:31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art. See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION ANDPURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998)18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154(1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263;Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997)190:139; Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles etal., J. BIOL. CHEM. (1996) 271(37):22376; Reverey et al., J. BIOL. CHEM.(1996) 271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270:4121;Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES(1993) 14(2):274. Commercially available liposomes include, for example,Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad, CA). Inaddition, calcium phosphate transfection may be used. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990)18(19):5667; and Mann and King. J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL. The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765).

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those of ordinary skill in the art. SeeMiller et al., BIOESSAYS (1989) 11(4):91; SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See Wright,NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56:153; Smithet al., MOL. CELL. BIOL. (1983) 3:2156. &e generally, Fraser et al., INVITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, the cell linesused for baculovirus expression vector systems commonly include, but arenot limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21(Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad,CA)), Tri-368 (Trichopulsia ni), and High-Five™ BTI-TN-5B1-4(Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those of ordinary skill in the art.

E. Coli, Pseudomonas Species, and Other Prokaryotes

Bacterial expression techniques are known to those of ordinary skill inthe art. A wide variety of vectors are available for use in bacterialhosts. The vectors may be single copy or low or high multicopy vectors.Vectors may serve for cloning and/or expression. In view of the ampleliterature concerning vectors, commercial availability of many vectors,and even manuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers is present, which providefor different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., NUC. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos. 036776 and 121 775, which are incorporated by reference herein]. Theβ-galactosidase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes.” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al., NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406, which are incorporated by reference herein]promoter systems also provide useful promoter sequences. Preferredmethods of the present invention utilize strong promoters, such as theT7 promoter to induce relaxin polypeptides at high levels. Examples ofsuch vectors are known to those of ordinary skill in the art and includethe pET29 series from Novagen, and the pPOP vectors described inWO99/05297, which is incorporated by reference herein. Such expressionsystems produce high levels of relaxin polypeptides in the host withoutcompromising host cell viability or growth parameters. pET19 (Novagen)is another vector known in the art.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EP Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding a relaxin polypeptide, are included in theprogeny intended by this definition.

The selection of suitable host bacteria for expression of relaxinpolypeptides is known to those of ordinary skill in the art. Inselecting bacterial hosts for expression, suitable hosts may includethose shown to have, inter alia, good inclusion body formation capacity,low proteolytic activity, and overall robustness. Bacterial hosts aregenerally available from a variety of sources including, but not limitedto, the Bacterial Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, CA); and theAmerican Type Culture Collection (“ATCC”) (Manassas, VA).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. Other examples of suitable E. coli hostsinclude, but are not limited to, strains of BL21, DH10B, or derivativesthereof. In another embodiment of the methods of the present invention,the E. coli host is a protease minus strain including, but not limitedto, OMP− and LON−. The host cell strain may be a species of Pseudomonas,including but not limited to, Pseudomonas fluorescens, Pseudomonasaeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biovar 1,designated strain MB101, is known to be useful for recombinantproduction and is available for therapeutic protein productionprocesses. Examples of a Pseudomonas expression system include thesystem available from The Dow Chemical Company as a host strain(Midland, MI available on the World Wide Web at dow.com).

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of relaxin polypeptides. As will be apparent to one ofskill in the art, the method of culture of the recombinant host cellstrain will be dependent on the nature of the expression constructutilized and the identity of the host cell. Recombinant host strains arenormally cultured using methods that are known to those of ordinaryskill in the art. Recombinant host cells are typically cultured inliquid medium containing assimilatable sources of carbon, nitrogen, andinorganic salts and, optionally, containing vitamins, amino acids,growth factors, and other proteinaceous culture supplements known tothose of ordinary skill in the art. Liquid media for culture of hostcells may optionally contain antibiotics or anti-fungals to prevent thegrowth of undesirable microorganisms and/or compounds including, but notlimited to, antibiotics to select for host cells containing theexpression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the relaxin polypeptideaccumulates intracellularly) or harvesting of culture supernatant ineither batch or continuous formats. For production in prokaryotic hostcells, batch culture and cell harvest are preferred.

The relaxin polypeptides of the present invention are normally purifiedafter expression in recombinant systems. The relaxin polypeptide may bepurified from host cells or culture medium by a variety of methods knownto the art. Relaxin polypeptides produced in bacterial host cells may bepoorly soluble or insoluble (in the form of inclusion bodies). In oneembodiment of the present invention, amino acid substitutions mayreadily be made in the relaxin polypeptide that are selected for thepurpose of increasing the solubility of the recombinantly producedprotein utilizing the methods disclosed herein as well as those known inthe art. In the case of insoluble protein, the protein may be collectedfrom host cell lysates by centrifugation and may further be followed byhomogenization of the cells. In the case of poorly soluble protein,compounds including, but not limited to, polyethylene imine (PEI) may beadded to induce the precipitation of partially soluble protein. Theprecipitated protein may then be conveniently collected bycentrifugation. Recombinant host cells may be disrupted or homogenizedto release the inclusion bodies from within the cells using a variety ofmethods known to those of ordinary skill in the art. Host celldisruption or homogenization may be performed using well knowntechniques including, but not limited to, enzymatic cell disruption,sonication, dounce homogenization, or high pressure release disruption.In one embodiment of the method of the present invention, the highpressure release technique is used to disrupt the E. coli host cells torelease the inclusion bodies of the relaxin polypeptides. When handlinginclusion bodies of relaxin polypeptide, it may be advantageous tominimize the homogenization time on repetitions in order to maximize theyield of inclusion bodies without loss due to factors such assolubilization, mechanical shearing or proteolysis.

Insoluble or precipitated relaxin polypeptide may then be solubilizedusing any of a number of suitable solubilization agents known to theart. The relaxin polypeptide may be solubilized with urea or guanidinehydrochloride. The volume of the solubilized relaxin polypeptide shouldbe minimized so that large batches may be produced using convenientlymanageable batch sizes. This factor may be significant in a large-scalecommercial setting where the recombinant host may be grown in batchesthat are thousands of liters in volume. In addition, when manufacturingrelaxin polypeptide in a large-scale commercial setting, in particularfor human pharmaceutical uses, the avoidance of harsh chemicals that candamage the machinery and container, or the protein product itself,should be avoided, if possible. It has been shown in the method of thepresent invention that the milder denaturing agent urea can be used tosolubilize the relaxin polypeptide inclusion bodies in place of theharsher denaturing agent guanidine hydrochloride. The use of ureasignificantly reduces the risk of damage to stainless steel equipmentutilized in the manufacturing and purification process of relaxinpolypeptide while efficiently solubilizing the relaxin polypeptideinclusion bodies.

In the case of soluble relaxin protein, the relaxin may be secreted intothe periplasmic space or into the culture medium. In addition, solublerelaxin may be present in the cytoplasm of the host cells. It may bedesired to concentrate soluble relaxin prior to performing purificationsteps. Standard techniques known to those of ordinary skill in the artmay be used to concentrate soluble relaxin from, for example, celllysates or culture medium. In addition, standard techniques known tothose of ordinary skill in the art may be used to disrupt host cells andrelease soluble relaxin from the cytoplasm or periplasmic space of thehost cells.

When relaxin polypeptide is produced as a fusion protein, the fusionsequence may be removed. Removal of a fusion sequence may beaccomplished by enzymatic or chemical cleavage. Enzymatic removal offusion sequences may be accomplished using methods known to those ofordinary skill in the art. The choice of enzyme for removal of thefusion sequence will be determined by the identity of the fusion, andthe reaction conditions will be specified by the choice of enzyme aswill be apparent to one of ordinary skill in the art. Chemical cleavagemay be accomplished using reagents known to those of ordinary skill inthe art, including but not limited to, cyanogen bromide, TEV protease,and other reagents. The cleaved relaxin polypeptide may be purified fromthe cleaved fusion sequence by methods known to those of ordinary skillin the art. Such methods will be determined by the identity andproperties of the fusion sequence and the relaxin polypeptide, as willbe apparent to one of ordinary skill in the art. Methods forpurification may include, but are not limited to, size-exclusionchromatography, hydrophobic interaction chromatography, ion-exchangechromatography or dialysis or any combination thereof.

The relaxin polypeptide may also be purified to remove DNA from theprotein solution. DNA may be removed by any suitable method known to theart, such as precipitation or ion exchange chromatography, but may beremoved by precipitation with a nucleic acid precipitating agent, suchas, but not limited to, protamine sulfate. The relaxin polypeptide maybe separated from the precipitated DNA using standard well known methodsincluding, but not limited to, centrifugation or filtration. Removal ofhost nucleic acid molecules is an important factor in a setting wherethe relaxin polypeptide is to be used to treat humans and the methods ofthe present invention reduce host cell DNA to pharmaceuticallyacceptable levels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human relaxin polypeptides of the invention can generally be recoveredusing methods standard in the art. For example, culture medium or celllysate can be centrifuged or filtered to remove cellular debris. Thesupernatant may be concentrated or diluted to a desired volume ordiafiltered into a suitable buffer to condition the preparation forfurther purification. Further purification of the relaxin polypeptide ofthe present invention includes separating deamidated and clipped formsof the relaxin polypeptide variant from the intact form.

Any of the following exemplary procedures can be employed forpurification of relaxin polypeptides of the invention: affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;high performance liquid chromatography (HPLC); reverse phase HPLC; gelfiltration (using, including but not limited to, SEPHADEX G-75);hydrophobic interaction chromatography; size-exclusion chromatography;metal-chelate chromatography; ultrafiltration/diafiltration; ethanolprecipitation; ammonium sulfate precipitation; chromatofocusing;displacement chromatography; electrophoretic procedures (including butnot limited to preparative isoelectric focusing), differentialsolubility (including but not limited to ammonium sulfateprecipitation), SDS-PAGE, or extraction.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, peptides comprising unnaturalamino acids, antibodies to proteins comprising unnatural amino acids,binding partners for proteins comprising unnatural amino acids, etc.,can be purified, either partially or substantially to homogeneity,according to standard procedures known to and used by those of skill inthe art. Accordingly, polypeptides of the invention can be recovered andpurified by any of a number of methods known to those of ordinary skillin the art, including but not limited to, ammonium sulfate or ethanolprecipitation, acid or base extraction, column chromatography, affinitycolumn chromatography, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography, lectin chromatography, gelelectrophoresis and the like. Protein refolding steps can be used, asdesired, in making correctly folded mature proteins. High performanceliquid chromatography (HPLC), affinity chromatography or other suitablemethods can be employed in final purification steps where high purity isdesired. In one embodiment, antibodies made against unnatural aminoacids (or proteins or peptides comprising unnatural amino acids) areused as purification reagents, including but not limited to, foraffinity-based purification of proteins or peptides comprising one ormore unnatural amino acid(s). Once purified, partially or tohomogeneity, as desired, the polypeptides are optionally used for a widevariety of utilities, including but not limited to, as assay components,therapeutics, prophylaxis, diagnostics, research reagents, and/or asimmunogens for antibody production. Antibodies generated againstpolypeptides of the present invention may be obtained by administeringthe polypeptides or epitope-bearing fragments, or cells to an animal,preferably a non-human animal, using routine protocols. One of ordinaryskill in the art could generate antibodies using a variety of knowntechniques. Also, transgenic mice, or other organisms, including othermammals, may be used to express humanized antibodies. Theabove-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides.Antibodies against polypeptides of the present invention may also beemployed to treat diseases.

Polypeptides and polynucleotides of the present invention may also beused as vaccines. Accordingly, in a further aspect, the presentinvention relates to a method for inducing an immunological response ina mammal that comprises inoculating the mammal with a polypeptide of thepresent invention, adequate to produce antibody and/or T cell immuneresponse, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said animal from disease, whether thatdisease is already established within the individual or not. Animmunological response in a mammal may also be induced by a methodcomprises delivering a polypeptide of the present invention via a vectordirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect said animal from diseases of the invention.One way of administering the vector is by accelerating it into thedesired cells as a coating on particles or otherwise. Such nucleic acidvector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNAhybrid. For use as a vaccine, a polypeptide or a nucleic acid vectorwill be normally provided as a vaccine formulation (composition). Theformulation may further comprise a suitable carrier. Since a polypeptidemay be broken down in the stomach, it may be administered parenterally(for instance, subcutaneous, intramuscular, intravenous, or intra-dermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions that may containanti-oxidants, buffers, bacteriostats and solutes that render theformulation instonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions that may include suspending agents orthickening agents. The vaccine formulation may also include adjuvantsystems for enhancing the immunogenicity of the formulation which areknown to those of ordinary skill in the art. The dosage will depend onthe specific activity of the vaccine and can be readily determined byroutine experimentation.

Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe Relaxin polypeptides of the present invention. Derivatization ofamino acids with reactive side-chains such as Lys, Cys and Tyr resultedin the conversion of lysine to N²-acetyl-lysine. Chemical synthesis alsoprovides a straightforward method to incorporate unnatural amino acids.With the recent development of enzymatic ligation and native chemicalligation of peptide fragments, it is possible to make larger proteins.See, e.g., P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem, 69:923(2000). Chemical peptide ligation and native chemical ligation aredescribed in U.S. Pat. No. 6,184,344, U.S. Patent Publication No.2004/0138412, U.S. Patent Publication No. 2003/0208046, WO 02/098902,and WO 03/042235, which are incorporated by reference herein. A generalin vitro biosynthetic method in which a suppressor tRNA chemicallyacylated with the desired unnatural amino acid is added to an in vitroextract capable of supporting protein biosynthesis, has been used tosite-specifically incorporate over 100 unnatural amino acids into avariety of proteins of virtually any size. See, e.g., V. W. Cornish, D.Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995, 34:621(1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244:182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111:8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction.

In addition to other references noted herein, a variety ofpurification/protein folding methods are known to those of ordinaryskill in the art, including, but not limited to, those set forth in R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification, AcademicPress, Inc. N.Y. (1990); Sandana, (1997) Bioseparation of Proteins,Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd EditionWiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal, (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal, Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes, (1993) Protein Purification: Principlesand Practice 3rd Edition Springer Verlag, NY; Janson and Ryden, (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998), ProteinProtocols on CD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins or peptides canpossess a conformation different from the desired conformations of therelevant polypeptides. In one aspect of the invention, the expressedprotein or polypeptide is optionally denatured and then renatured. Thisis accomplished utilizing methods known in the art, including but notlimited to, by adding a chaperonin to the protein or polypeptide ofinterest, by solubilizing the proteins in a chaotropic agent such asguanidine HCl, utilizing protein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are known tothose of ordinary skill in the art (see, the references above, andDebinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman andPastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992)Anal. Biochem., 205: 263-270). Debinski, et al., for example, describethe denaturation and reduction of inclusion body proteins inguanidine-DTE. The proteins can be refolded in a redox buffercontaining, including but not limited to, oxidized glutathione andL-arginine. Refolding reagents can be flowed or otherwise moved intocontact with the one or more polypeptide or other expression product, orvice-versa.

In the case of prokaryotic production of relaxin polypeptide, therelaxin polypeptide thus produced may be misfolded and thus lacks or hasreduced biological activity. The bioactivity of the protein may berestored by “refolding”. In general, misfolded relaxin polypeptide isrefolded by solubilizing (where the relaxin polypeptide is alsoinsoluble), unfolding and reducing the polypeptide chain using, forexample, one or more chaotropic agents (e.g. urea and/or guanidine) anda reducing agent capable of reducing disulfide bonds (e.g.dithiothreitol, DTT or 2-mercaptoethanol, 2-ME). At a moderateconcentration of chaotrope, an oxidizing agent is then added (e.g.,oxygen, cystine or cystamine), which allows the reformation of disulfidebonds. Relaxin polypeptide may be refolded using standard methods knownin the art, such as those described in U.S. Pat. Nos. 4,511,502,4,511,503, and 4,512,922, which are incorporated by reference herein.The relaxin polypeptide may also be cofolded with other proteins to formheterodimers or heteromultimers.

After refolding, the relaxin may be further purified. Purification ofrelaxin may be accomplished using a variety of techniques known to thoseof ordinary skill in the art, including hydrophobic interactionchromatography, size exclusion chromatography, ion exchangechromatography, reverse-phase high performance liquid chromatography,affinity chromatography, and the like or any combination thereof.Additional purification may also include a step of drying orprecipitation of the purified protein.

After purification, relaxin may be exchanged into different buffersand/or concentrated by any of a variety of methods known to the art,including, but not limited to, diafiltration and dialysis. Relaxin thatis provided as a single purified protein may be subject to aggregationand precipitation.

The purified relaxin may be at least 90% pure (as measured by reversephase high performance liquid chromatography, RP-HPLC, or sodium dodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or at least 95%pure, or at least 98% pure, or at least 99% or greater pure. Regardlessof the exact numerical value of the purity of the relaxin, the relaxinis sufficiently pure for use as a pharmaceutical product or for furtherprocessing, such as conjugation with a water soluble polymer such asPEG.

Certain relaxin molecules may be used as therapeutic agents in theabsence of other active ingredients or proteins (other than excipients,carriers, and stabilizers, serum albumin and the like), or they may becomplexed with another protein or a polymer.

General Purification Methods

Any one of a variety of isolation steps may be performed on the celllysate, extract, culture medium, inclusion bodies, periplasmic space ofthe host cells, cytoplasm of the host cells, or other material,comprising relaxin polypeptide or on any relaxin polypeptide mixturesresulting from any isolation steps including, but not limited to,affinity chromatography, ion exchange chromatography, hydrophobicinteraction chromatography, gel filtration chromatography, highperformance liquid chromatography (“HPLC”), reversed phase-HPLC(“RP-HPLC”), expanded bed adsorption, or any combination and/orrepetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, CA), Bio-Rad Laboratories,Inc. (Hercules, CA), and Amersham Biosciences, Inc. (Piscataway, NJ).Chromatographic materials including, but not limited to, exchange matrixmaterials, media, and buffers are also available from such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, NJ).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, NJ). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, NJ).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway,NJ). Indeed, entire systems are commercially available including thevarious AKTA® systems from Amersham Biosciences (Piscataway, NJ).

In one embodiment of the present invention, for example, the relaxinpolypeptide may be reduced and denatured by first denaturing theresultant purified relaxin polypeptide in urea, followed by dilutioninto TRIS buffer containing a reducing agent (such as DTT) at a suitablepH. In another embodiment, the relaxin polypeptide is denatured in ureain a concentration range of between about 2 M to about 9 M, followed bydilution in TRIS buffer at a pH in the range of about 5.0 to about 8.0.The refolding mixture of this embodiment may then be incubated. In oneembodiment, the refolding mixture is incubated at room temperature forfour to twenty-four hours. The reduced and denatured relaxin polypeptidemixture may then be further isolated or purified.

As stated herein, the pH of the first relaxin polypeptide mixture may beadjusted prior to performing any subsequent isolation steps. Inaddition, the first relaxin polypeptide mixture or any subsequentmixture thereof may be concentrated using techniques known in the art.Moreover, the elution buffer comprising the first relaxin polypeptidemixture or any subsequent mixture thereof may be exchanged for a buffersuitable for the next isolation step using techniques known to those ofordinary skill in the art.

Ion Exchange Chromatography

In one embodiment, and as an optional, additional step, ion exchangechromatography may be performed on the first relaxin polypeptidemixture. See generally ION EXCHANGE CHROMATOGRAPHY: PRINCIPLES ANDMETHODS (Cat. No. 18-1114-21, Amersham Biosciences (Piscataway, NJ)).Commercially available ion exchange columns include HITRAP®, HIPREP, andHILOAD® Columns (Amersham Biosciences, Piscataway, NJ). Such columnsutilize strong anion exchangers such as Q SEPHAROSE® Fast Flow, QSEPHAROSE® High Performance, and Q SEPHAROSE® XL; strong cationexchangers such as SP SEPHAROSE® High Performance, SP SEPHAROSE® FastFlow, and SP SEPHAROSE® XL; weak anion exchangers such as DEAESEPHAROSE® Fast Flow; and weak cation exchangers such as CM SEPHAROSE®Fast Flow (Amersham Biosciences, Piscataway, NJ). Anion or cationexchange column chromatography may be performed on the relaxinpolypeptide at any stage of the purification process to isolatesubstantially purified relaxin polypeptide. The cation exchangechromatography step may be performed using any suitable cation exchangematrix. Useful cation exchange matrices include, but are not limited to,fibrous, porous, non-porous, microgranular, beaded, or cross-linkedcation exchange matrix materials. Such cation exchange matrix materialsinclude, but are not limited to, cellulose, agarose, dextran,polyacrylate, polyvinyl, polystyrene, silica, polyether, or compositesof any of the foregoing.

The cation exchange matrix may be any suitable cation exchangerincluding strong and weak cation exchangers. Strong cation exchangersmay remain ionized over a wide pH range and thus, may be capable ofbinding relaxin over a wide pH range. Weak cation exchangers, however,may lose ionization as a function of pH. For example, a weak cationexchanger may lose charge when the pH drops below about pH 4 or pH 5.Suitable strong cation exchangers include, but are not limited to,charged functional groups such as sulfopropyl (SP), methyl sulfonate(S), or sulfoethyl (SE). The cation exchange matrix may be a strongcation exchanger, preferably having a relaxin binding pH range of about2.5 to about 6.0. Alternatively, the strong cation exchanger may have arelaxin binding pH range of about pH 2.5 to about pH 5.5. The cationexchange matrix may be a strong cation exchanger having a relaxinbinding pH of about 3.0. Alternatively, the cation exchange matrix maybe a strong cation exchanger, preferably having a relaxin binding pHrange of about 6.0 to about 8.0. The cation exchange matrix may be astrong cation exchanger preferably having a relaxin binding pH range ofabout 8.0 to about 12.5. Alternatively, the strong cation exchanger mayhave a relaxin binding pH range of about pH 8.0 to about pH 12.0.

Prior to loading the relaxin, the cation exchange matrix may beequilibrated, for example, using several column volumes of a dilute,weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.Following equilibration, the relaxin may be added and the column may bewashed one to several times, prior to elution of substantially purifiedrelaxin, also using a weak acid solution such as a weak acetic acid orphosphoric acid solution. For example, approximately 2-4 column volumesof 20 mM acetic acid, pH 3, may be used to wash the column. Additionalwashes using, e.g., 2-4 column volumes of 0.05 M sodium acetate, pH 5.5,or 0.05 M sodium acetate mixed with 0.1 M sodium chloride, pH 5.5, mayalso be used. Alternatively, using methods known in the art, the cationexchange matrix may be equilibrated using several column volumes of adilute, weak base.

Alternatively, substantially purified relaxin may be eluted bycontacting the cation exchanger matrix with a buffer having asufficiently low pH or ionic strength to displace the relaxin from thematrix. The pH of the elution buffer may range from about pH 2.5 toabout pH 6.0. More specifically, the pH of the elution buffer may rangefrom about pH 2.5 to about pH 5.5, about pH 2.5 to about pH 5.0. Theelution buffer may have a pH of about 3.0. In addition, the quantity ofelution buffer may vary widely and will generally be in the range ofabout 2 to about 10 column volumes.

Following adsorption of the relaxin polypeptide to the cation exchangermatrix, substantially purified relaxin polypeptide may be eluted bycontacting the matrix with a buffer having a sufficiently high pH orionic strength to displace the relaxin polypeptide from the matrix.Suitable buffers for use in high pH elution of substantially purifiedrelaxin polypeptide may include, but not limited to, citrate, phosphate,formate, acetate, HEPES, and MES buffers ranging in concentration fromat least about 5 mM to at least about 100 mM.

Reverse-Phase Chromatography

RP-HPLC may be performed to purify proteins following suitable protocolsthat are known to those of ordinary skill in the art. See, e.g., Pearsonet al., ANAL BIOCHEM. (1982) 124:217-230 (1982); Rivier et al., J.CHROM. (1983) 268:112-119; Kunitani et al., J. CHROM. (1986)359:391-402. RP-HPLC may be performed on the relaxin polypeptide toisolate substantially purified relaxin polypeptide. In this regard,silica derivatized resins with alkyl functionalities with a wide varietyof lengths, including, but not limited to, at least about C3 to at leastabout C30, at least about C3 to at least about C20, or at least about C3to at least about C18, resins may be used. Alternatively, a polymericresin may be used. For example, TosoHaas Amberchrome CG1000sd resin maybe used, which is a styrene polymer resin. Cyano or polymeric resinswith a wide variety of alkyl chain lengths may also be used.Furthermore, the RP-HPLC column may be washed with a solvent such asethanol. The Source RP column is another example of a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the relaxin polypeptide from the RP-HPLCcolumn. The most commonly used ion pairing agents include, but are notlimited to, acetic acid, formic acid, perchloric acid, phosphoric acid,trifluoroacetic acid, heptafluorobutyric acid, triethylamine,tetramethylammonium, tetrabutylammonium, and triethylammonium acetate.Elution may be performed using one or more gradients or isocraticconditions, with gradient conditions preferred to reduce the separationtime and to decrease peak width. Another method involves the use of twogradients with different solvent concentration ranges. Examples ofsuitable elution buffers for use herein may include, but are not limitedto, ammonium acetate and acetonitrile solutions.

Hydrophobic Interaction Chromatography Purification Techniques

Hydrophobic interaction chromatography (HIC) may be performed on therelaxin polypeptide. See generally HYDROPHOBIC INTERACTIONCHROMATOGRAPHY HANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90,Amersham Biosciences (Piscataway, NJ) which is incorporated by referenceherein. Suitable HIC matrices may include, but are not limited to,alkyl- or aryl-substituted matrices, such as butyl-, hexyl-, octyl- orphenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, NJ).

Briefly, prior to loading, the HIC column may be equilibrated usingstandard buffers known to those of ordinary skill in the art, such as anacetic acid/sodium chloride solution or HEPES containing ammoniumsulfate. Ammonium sulfate may be used as the buffer for loading the HICcolumn. After loading the relaxin polypeptide, the column may thenwashed using standard buffers and conditions to remove unwantedmaterials but retaining the relaxin polypeptide on the HIC column. Therelaxin polypeptide may be eluted with about 3 to about 10 columnvolumes of a standard buffer, such as a HEPES buffer containing EDTA andlower ammonium sulfate concentration than the equilibrating buffer, oran acetic acid/sodium chloride buffer, among others. A decreasing linearsalt gradient using, for example, a gradient of potassium phosphate, mayalso be used to elute the relaxin molecules. The eluant may then beconcentrated, for example, by filtration such as diafiltration orultrafiltration. Diafiltration may be utilized to remove the salt usedto elute the relaxin polypeptide.

Other Purification Techniques

Yet another isolation step using, for example, gel filtration (GELFILTRATION: PRINCIPLES AND METHODS (Cat. No. 18-1022-18, AmershamBiosciences, Piscataway, NJ) which is incorporated by reference herein,hydroxyapatite chromatography (suitable matrices include, but are notlimited to, HA-Ultrogel, High Resolution (Calbiochem), CHT CeramicHydroxyapatite (BioRad), Bio-Gel HTP Hydroxyapatite (BioRad)), HPLC,expanded bed adsorption, ultrafiltration, diafiltration, hyophilization,and the like, may be performed on the first relaxin polypeptide mixtureor any subsequent mixture thereof, to remove any excess salts and toreplace the buffer with a suitable buffer for the next isolation step oreven formulation of the final drug product.

The yield of relaxin polypeptide, including substantially purifiedrelaxin polypeptide, may be monitored at each step described hereinusing techniques known to those of ordinary skill in the art. Suchtechniques may also be used to assess the yield of substantiallypurified relaxin polypeptide following the last isolation step. Forexample, the yield of relaxin polypeptide may be monitored using any ofseveral reverse phase high pressure liquid chromatography columns,having a variety of alkyl chain lengths such as cyano RP-HPLC,C18RP-HPLC; as well as cation exchange HPLC and gel filtration HPLC.

In specific embodiments of the present invention, the yield of relaxinafter each purification step may be at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, at least about99.9%, or at least about 99.99%, of the relaxin in the starting materialfor each purification step.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring relaxin polypeptide using Western blot and ELISA assays.For example, polyclonal antibodies may be generated against proteinsisolated from negative control yeast fermentation and the cationexchange recovery. The antibodies may also be used to probe for thepresence of contaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of relaxinpolypeptide from the proteinaceous impurities is based on differences inthe strength of hydrophobic interactions. Elution is performed with anacetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLCis performed using a stainless steel column (filled with 2.8 to 3.2liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate isacidified by adding trifluoroacetic acid and loaded onto the Vydac C4column. For washing and elution an acetonitrile gradient in dilutedtrifluoroacetic acid is used. Fractions are collected and immediatelyneutralized with phosphate buffer. The relaxin polypeptide fractionswhich are within the IPC limits are pooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of relaxin polypeptide to the DEAE groups is mediatedby ionic interactions. Acetonitrile and trifluoroacetic acid passthrough the column without being retained. After these substances havebeen washed off, trace impurities are removed by washing the column withacetate buffer at a low pH. Then the column is washed with neutralphosphate buffer and relaxin polypeptide is eluted with a buffer withincreased ionic strength. The column is packed with DEAE Sepharose fastflow. The column volume is adjusted to assure a relaxin polypeptide loadin the range of 3-10 mg relaxin polypeptide/ml gel. The column is washedwith water and equilibration buffer (sodium/potassium phosphate). Thepooled fractions of the HPLC eluate are loaded and the column is washedwith equilibration buffer. Then the column is washed with washing buffer(sodium acetate buffer) followed by washing with equilibration buffer.Subsequently, relaxin polypeptide is eluted from the column with elutionbuffer (sodium chloride, sodium/potassium phosphate) and collected in asingle fraction in accordance with the master elution profile. Theeluate of the DEAE Sepharose column is adjusted to the specifiedconductivity. The resulting drug substance is sterile filtered intoTeflon bottles and stored at −70° C.

Additional methods that may be employed include, but are not limited to,steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs)which are located on the outer membrane of Gram-negative host cells,such as, for example, Escherichia coli. Methods for reducing endotoxinlevels are known to one of ordinary skill in the art and include, butare not limited to, purification techniques using silica supports, glasspowder or hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest. Methods for measuringendotoxin levels are known to one of ordinary skill in the art andinclude, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.The Endosafe™-PTS assay is a colorimetric, single tube system thatutilizes cartridges preloaded with LAL reagent, chromogenic substrate,and control standard endotoxin along with a handheld spectrophotometer.Alternate methods include, but are not limited to, a Kinetic LAL methodthat is turbidmetric and uses a 96 well format.

A wide variety of methods and procedures can be used to assess the yieldand purity of a relaxin protein comprising one or more non-naturallyencoded amino acids, including but not limited to, the Bradford assay,SDS-PAGE, silver stained SDS-PAGE, coomassie stained SDS-PAGE, massspectrometry (including but not limited to, MALDI-TOF) and other methodsfor characterizing proteins known to one of ordinary skill in the art.

Additional methods include, but are not limited to: SDS-PAGE coupledwith protein staining methods, immunoblotting, matrix assisted laserdesorption/ionization-mass spectrometry (MALDI-MS), liquidchromatography/mass spectrometry, isoelectric focusing, analytical anionexchange, chromatofocusing, and circular dichroism.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle. J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. van Hest and D. A. Tirrell,FEBS Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A.Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S.Patent Publication 2002/0042097, which are incorporated by referenceherein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS. Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn. H. Studies on polypeptides.XXXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enzymes, Ace ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson. D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenzyme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J Biol. Chem,243(24):6392-6401 (1968); Polgar, L. et M. L. Bender. A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. AmChem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.Schultz, P. G. Introduction of nucleophiles and spectroscopic probesinto antibody combining sites, Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill. S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz. P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041):197-200 (1992).

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987.Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded the repertoire ofcatalysis to various chemical reactions. Studies have identified RNAmolecules that can catalyze aminoacyl-RNA bonds on their own(2)3′-termini (Illangakekare et al., 1995 Science 267:643-647), and anRNA molecule which can transfer an amino acid from one RNA molecule toanother (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturallyencoded amino acids. Substrate-immobilized forms of enzymatic moleculesthat can aminoacylate tRNAs, including but not limited to, ribozymes,may enable efficient affinity purification of the aminoacylatedproducts. Examples of suitable substrates include agarose, sepharose,and magnetic beads. The production and use of a substrate-immobilizedform of ribozyme for aminoacylation is described in Chemistry andBiology 2003, 10:1077-1084 and U.S. Patent Application Publication2003/0228593, which are incorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty. D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), whichare incorporated by reference herein, to avoid the use of synthetases inaminoacylation. Such methods or other chemical aminoacylation methodsmay be used to aminoacylate tRNA molecules.

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24mini conjugated with tRNA^(Asn) _(CCCG), followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Immobilization on a substrate may be used to enable efficient affinitypurification of the aminoacylated tRNAs. Examples of suitable substratesinclude, but are not limited to, agarose, sepharose, and magnetic beads.Ribozymes can be immobilized on resins by taking advantage of thechemical structure of RNA, such as the 3′-cis-diol on the ribose of RNAcan be oxidized with periodate to yield the corresponding dialdehyde tofacilitate immobilization of the RNA on the resin. Various types ofresins can be used including inexpensive hydrazide resins whereinreductive amination makes the interaction between the resin and theribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can besignificantly facilitated by this on-column aminoacylation technique.Kourouklis et al. Methods 2005; 36:239-4 describe a column-basedaminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAs of thepresent invention may be used include, but are not limited to celllysates. Cell lysates provide reaction components necessary for in vitrotranslation of a polypeptide from an input mRNA. Examples of suchreaction components include but are not limited to ribosomal proteins,rRNA, amino acids, tRNAs, GTP, ATP, translation initiation andelongation factors and additional factors associated with translation.Additionally, translation systems may be batch translations orcompartmentalized translation. Batch translation systems combinereaction components in a single compartment while compartmentalizedtranslation systems separate the translation reaction components fromreaction products that can inhibit the translation efficiency. Suchtranslation systems are available commercially.

Further, a coupled transcription/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.). The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Stephan in Scientist 2005 Oct. 10; pages 30-33 describes additionalmethods to incorporate non-naturally encoded amino acids into proteins.Lu et al. in Mol Cell. 2001 October; 8(4):759-69 describe a method inwhich a protein is chemically ligated to a synthetic peptide containingunnatural amino acids (expressed protein ligation).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty. Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman. P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers a wide variety of advantages including but not limitedto, high yields of mutant proteins, technical ease, the potential tostudy the mutant proteins in cells or possibly in living organisms andthe use of these mutant proteins in therapeutic treatments anddiagnostic uses. The ability to include unnatural amino acids withvarious sizes, acidities, nucleophilicities, hydrophobicities, and otherproperties into proteins can greatly expand our ability to rationallyand systematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998).

It may also be possible to obtain expression of a relaxin polynucleotideof the present invention using a cell-free (in-vitro) translationalsystem. Translation systems may be cellular or cell-free, and may beprokaryotic or eukaryotic. Cellular translation systems include, but arenot limited to, whole cell preparations such as permeabilized cells orcell cultures wherein a desired nucleic acid sequence can be transcribedto mRNA and the mRNA translated. Cell-free translation systems amcommercially available and many different types and systems arewell-known. Examples of cell-free systems include, but are not limitedto, prokaryotic lysates such as Escherichia coli lysates, and eukaryoticlysates such as wheat germ extracts, insect cell lysates, rabbitreticulocyte lysates, rabbit oocyte lysates and human cell lysates.Eukaryotic extracts or lysates may be preferred when the resultingprotein is glycosylated, phosphorylated or otherwise modified becausemany such modifications are only possible in eukaryotic systems. Some ofthese extracts and lysates are available commercially (Promega; Madison,Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, Ill.;GIBCO/BRL; Grand Island, N.Y.). Membranous extracts, such as the caninepancreatic extracts containing microsomal membranes, are also availablewhich are useful for translating secretory proteins. In these systems,which can include either mRNA as a template (in-vitro translation) orDNA as a template (combined in-vitro transcription and translation), thein vitro synthesis is directed by the ribosomes. Considerable effort hasbeen applied to the development of cell-free protein expression systems.See, e.g., Kim, D. M, and J. R. Swartz, Biotechnology andBioengineering, 74:309-316 (2001): Kim, D. M, and J. R. Swartz,Biotechnology Letters, 22, 1537-1542, (2000); Kim, D. M., and J. R.Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D. M., and J.R. Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999); andPatnaik, R, and J. R. Swartz, Biotechniques 24, 862-868, (1998); U.S.Pat. No. 6,337,191: U.S. Patent Publication No. 2002/0081660; WO00/55353; WO 90/05785, which are incorporated by reference herein.Another approach that may be applied to the expression of relaxinpolypeptides comprising a non-naturally encoded amino acid includes themRNA-peptide fusion technique. See, e.g., R. Roberts and J. Szostak,Proc. Natl Acad. Sci. (USA) 94:12297-12302 (1997); A. Frankel, et al.,Chemistry & Biology 10:1043-1050 (2003). In this approach, an mRNAtemplate linked to puromycin is translated into peptide on the ribosome.If one or more tRNA molecules has been modified, non-natural amino acidscan be incorporated into the peptide as well. After the last mRNA codonhas been read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of relaxin polypeptidescomprising one or more non-naturally encoded amino acids to identifypolypeptides having desired properties. More recently, in vitro ribosometranslations with purified components have been reported that permit thesynthesis of peptides substituted with non-naturally encoded aminoacids. See, e.g., A. Forster et al., Proc. Natl Acad. Sci. (USA)100:6353 (2003).

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

Macromolecular Polymers Coupled to Relaxin Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide; a water-soluble dendrimer; acyclodextrin; an inhibitory ribonucleic acid; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety: anactinic radiation excitable moiety; a photoisomerizable moiety; biotin:a derivative of biotin; a biotin analogue; a moiety incorporating aheavy atom; a chemically cleavable group; a photocleavable group; anelongated side chain; a carbon-linked sugar; a redox-active agent; anamino thioacid; a toxic moiety; an isotopically labeled moiety; abiophysical probe; a phosphorescent group; a chemiluminescent group; anelectron dense group; a magnetic group; an intercalating group; achromophore; an energy transfer agent; a biologically active agent; adetectable label; a small molecule; a quantum dot; a nanotransmitter; aradionucleotide; a radiotransmitter; a neutron-capture agent; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to relaxin polypeptides of the present invention to modulatebiological properties of the relaxin polypeptide, and/or provide newbiological properties to the relaxin molecule. These macromolecularpolymers can be linked to the Relaxin polypeptide via a naturallyencoded amino acid, via a non-naturally encoded amino acid, or anyfunctional substituent of a natural or non-natural amino acid, or anysubstituent or functional group added to a natural or non-natural aminoacid. The molecular weight of the polymer may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. The molecular weight of the polymer may be between about 100 Daand about 100,000 Da, including but not limited to, 100,000 Da, 95,000Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da,60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da,7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da,900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100Da. In some embodiments, the molecular weight of the polymer is betweenabout 100 Da and about 50,000 Da. In some embodiments, the molecularweight of the polymer is between about 100 Da and about 40,000 Da. Insome embodiments, the molecular weight of the polymer is between about1,000 Da and about 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and about 40,000 Da.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated relaxin polypeptide preparations provided herein are thosewhich are homogenous enough to display the advantages of a homogenouspreparation, e.g., ease in clinical application in predictability of lotto lot pharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.For therapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

Examples of polymers include but are not limited to polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof; hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixturesthereof; and derivatives of the foregoing.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG: relaxin polypeptideconjugates, the term “therapeutically effective amount” refers to anamount which gives the desired benefit to a patient. The amount willvary from one individual to another and will depend upon a number offactors, including the overall physical condition of the patient and theunderlying cause of the condition to be treated. The amount of relaxinpolypeptide used for therapy gives an acceptable rate of change andmaintains desired response at a beneficial level. A therapeuticallyeffective amount of the present compositions may be readily ascertainedby one of ordinary skill in the art using publicly available materialsand procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods known to those of ordinary skill in the art(Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,pages 138-161). The term “PEG” is used broadly to encompass anypolyethylene glycol molecule, without regard to size or to modificationat an end of the PEG, and can be represented as linked to the relaxinpolypeptide by the formula:XO—(CH₂CHO)_(n)—CH₂CH₂—Y

where n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to a relaxin polypeptide via a naturally-occurring ornon-naturally encoded amino acid. For instance, Y may be an amide,carbamate or urea linkage to an amine group (including but not limitedto, the epsilon amine of lysine or the N-terminus) of the polypeptide.Alternatively, Y may be a maleimide linkage to a thiol group (includingbut not limited to, the thiol group of cysteine). Alternatively, Y maybe a linkage to a residue not commonly accessible via the 20 commonamino acids. For example, an azide group on the PEG can be reacted withan alkyne group on the Relaxin polypeptide to form a Huisgen [3+2]cycloaddition product. Alternatively, an alkyne group on the PEG can bereacted with an azide group present in a non-naturally encoded aminoacid to form a similar product. In some embodiments, a strongnucleophile (including but not limited to, hydrazine, hydrazide,hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketonegroup present in a non-naturally encoded amino acid to form a hydrazone,oxime or semicarbazone, as applicable, which in some cases can befurther reduced by treatment with an appropriate reducing agent.Alternatively, the strong nucleophile can be incorporated into theRelaxin polypeptide via a non-naturally encoded amino acid and used toreact preferentially with a ketone or aldehyde group present in thewater soluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). The molecular weight of PEG may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. PEG may be between about 100 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, PEG isbetween about 100 Da and about 50,000 Da. In some embodiments, PEG isbetween about 100 Da and about 40,000 Da. In some embodiments, PEG isbetween about 1,000 Da and about 40,000 Da. In some embodiments, PEG isbetween about 5,000 Da and about 40,000 Da. In some embodiments, PEG isbetween about 10,000 Da and about 40,000 Da. Branched chain PEGs,including but not limited to, PEG molecules with each chain having a MWranging from 1-100 kDa (including but not limited to, 1-50 kDa or 5-20kDa) can also be used. The molecular weight of each chain of thebranched chain PEG may be, including but not limited to, between about1,000 Da and about 100,000 Da or more. The molecular weight of eachchain of the branched chain PEG may be between about 1,000 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In someembodiments, the molecular weight of each chain of the branched chainPEG is between about 1,000 Da and about 50,000 Da. In some embodiments,the molecular weight of each chain of the branched chain PEG is betweenabout 1,000 Da and about 40,000 Da. In some embodiments, the molecularweight of each chain of the branched chain PEG is between about 5,000 Daand about 40,000 Da. In some embodiments, the molecular weight of eachchain of the branched chain PEG is between about 5,000 Da and about20,000 Da. A wide range of PEG molecules are described in, including butnot limited to, the Shearwater Polymers, Inc. catalog, NektarTherapeutics catalog, incorporated herein by reference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acids as described herein. If the non-naturally encoded amino acidcomprises an azide, then the PEG will typically contain either an alkynemoiety to effect formation of the [3+2] cycloaddition product or anactivated PEG species (i.e., ester, carbonate) containing a phosphinegroup to effect formation of the amide linkage. Alternatively, if thenon-naturally encoded amino acid comprises an alkyne, then the PEG willtypically contain an azide moiety to effect formation of the [3+2]Huisgen cycloaddition product. If the non-naturally encoded amino acidcomprises a carbonyl group, the PEG will typically comprise a potentnucleophile (including but not limited to, a hydrazide, hydrazine,hydroxylamine, or semicarbazide functionality) in order to effectformation of corresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the Relaxin polypeptide variant with a PEGderivative contains a chemical functionality that is reactive with thechemical functionality present on the side chain of the non-naturallyencoded amino acid.

The invention provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and poly(propylene glycol), are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to encompass and include all such molecules. The term PEGincludes, but is not limited to, poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, derivatized PEG,forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically. PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone. The molecularweight of PEG may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof PEG may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of PEG is between about 100 Da and about 50,000 Da. In someembodiments, the molecular weight of PEG is between about 100 Da andabout 40,000 Da. In some embodiments, the molecular weight of PEG isbetween about 1,000 Da and about 40,000 Da. In some embodiments, themolecular weight of PEG is between about 5,000 Da and about 40,000 Da.In some embodiments, the molecular weight of PEG is between about 10,000Da and about 40,000 Da.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462; 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-

It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the forms knownin the art including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can van, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da. Themolecular weight of each chain of the polymer backbone may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of each chainof the polymer backbone is between about 100 Da and about 50,000 Da. Insome embodiments, the molecular weight of each chain of the polymerbackbone is between about 100 Da and about 40,000 Da. In someembodiments, the molecular weight of each chain of the polymer backboneis between about 1,000 Da and about 40,000 Da. In some embodiments, themolecular weight of each chain of the polymer backbone is between about5,000 Da and about 40,000 Da. In some embodiments, the molecular weightof each chain of the polymer backbone is between about 10,000 Da andabout 40,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:X-A-POLY-B—N═N═N

wherein:

N═N═N is an azide moiety;

B is a linking moiety, which may be present or absent:

POLY is a water-soluble non-antigenic polymer;

A is a linking moiety, which may be present or absent and which may bethe same as B or different; and

X is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen orsulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462; 5,643,575; andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is merely illustrative, and that all linking moieties having thequalities described above are contemplated to be suitable for use in thepresent invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those of ordinary skill in the art, theselected X moiety should be compatible with the azide group so thatreaction with the azide group does not occur. The azide-containingpolymer derivatives may be homobifunctional, meaning that the secondfunctional group (i.e., X) is also an azide moiety, orheterobifunctional, meaning that the second functional group is adifferent functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Makromol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benrotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. JBiochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. Nos. 5,824,784, 5,252,714),maleimide (see, e.g., Goodson et al. Biotechnology (NY) 8:343 (1990),Romani et al. in Chemistry of Peptides and Proteins 2:29 (1984)), andKogan, Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see,e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g.,Sawhney et al., Macromolecules, 26:581 (1993)), vinylsulfone (see, e.g.,U.S. Pat. No. 5,900,461). All of the above references and patents areincorporated herein by reference.

In certain embodiments of the present invention, the polymer derivativesof the invention comprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—N═N═N

wherein:

X is a functional group as described above; and

n is about 20 to about 4000.

In another embodiment, the polymer derivatives of the invention comprisea polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—W—N═N═N

wherein:

W is an aliphatic or aromatic linker moiety comprising between 1-10carbon atoms;

n is about 20 to about 4000; and

X is a functional group as described above. m is between 1 and 10.

The azide-containing PEG derivatives of the invention can be prepared bya variety of methods known in the art and/or disclosed herein. In onemethod, shown below, a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da, the polymerbackbone having a first terminus bonded to a first functional group anda second terminus bonded to a suitable leaving group, is reacted with anazide anion (which may be paired with any of a number of suitablecounter-ions, including sodium, potassium, tert-butylammonium and soforth). The leaving group undergoes a nucleophilic displacement and isreplaced by the azide moiety, affording the desired azide-containing PEGpolymer.X-PEG-L+N₃′→X-PEG-N₃

As shown, a suitable polymer backbone for use in the present inventionhas the formula X-PEG-L, wherein PEG is poly(ethylene glycol) and X is afunctional group which does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups include,but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl,amine, aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone. Examples of suitable leaving groupsinclude, but are not limited to, chloride, bromide, iodide, mesylate,tresylate, and tosylate.

In another method for preparation of the azide-containing polymerderivatives of the present invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:X-PEG-M+N-linker-N═N═N→PG-X-PEG-linker-N═N═N

wherein:

PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; and

M is a functional group that is not reactive with the azidefunctionality but that will react efficiently and selectively with the Nfunctional group.

Examples of suitable functional groups include, but are not limited to,M being a carboxylic acid, carbonate or active ester if N is an amine; Mbeing a ketone if N is a hydrazide or aminooxy moiety; M being a leavinggroup if N is a nucleophile.

Purification of the crude product may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:BocHN-PEG-NH₂+HO₂C—(CH₂)₃—N═N═N

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure:X-A-POLY-B—C≡C—R

wherein:

R can be either H or an alkyl, alkene, alkyoxy, or aryl or substitutedaryl group;

B is a linking moiety, which may be present or absent;

POLY is a water-soluble non-antigenic polymer;

A is a linking moiety, which may be present or absent and which may bethe same as B or different; and

X is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen,or sulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462 and 5,643,575 andU.S. Pat. Appl. Publication 2003/014356, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is intended to be merely illustrative, and that a wide variety oflinking moieties having the qualities described above are contemplatedto be useful in the present invention.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another embodiment of the present invention, the polymer derivativescomprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—C≡CH

wherein:

X is a functional group as described above;

n is about 20 to about 4000; and

m is between 1 and 10.

Specific examples of each of the heterobifunctional PEG polymers areshown below.

The acetylene-containing PEG derivatives of the invention can beprepared using methods known to those of ordinary skill in the artand/or disclosed herein. In one method, a water soluble polymer backbonehaving an average molecular weight from about 800 Da to about 100,000Da, the polymer backbone having a first terminus bonded to a firstfunctional group and a second terminus bonded to a suitable nucleophilicgroup, is reacted with a compound that bears both an acetylenefunctionality and a leaving group that is suitable for reaction with thenucleophilic group on the PEG. When the PEG polymer bearing thenucleophilic moiety and the molecule bearing the leaving group arecombined, the leaving group undergoes a nucleophilic displacement and isreplaced by the nucleophilic moiety, affording the desiredacetylene-containing polymer.X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR′

As shown, a preferred polymer backbone for use in the reaction has theformula X-PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN2-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via annucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In another embodiment of the present invention, A is an aliphatic linkerof between 1-10 carbon atoms or a substituted aryl ring of between 6-14carbon atoms. X is a functional group which does not react with azidegroups and L is a suitable leaving group

In another method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

An exemplary reaction scheme is shown below:X-PEG-L+-C≡CR′→X-PEG-C≡CR′

wherein:

PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; and

R′ is either H, an alkyl, alkoxy, aryl or aryloxy group or a substitutedalkyl, alkoxyl, aryl or aryloxy group.

In the example above, the leaving group L should be sufficientlyreactive to undergo SN2-type displacement when contacted with asufficient concentration of the acetylene anion. The reaction conditionsrequired to accomplish SN2 displacement of leaving groups by acetyleneanions are known to those of ordinary skill in the art.

Purification of the crude product can usually be accomplished by methodsknown in the art including, but are not limited to, precipitation of theproduct followed by chromatography, if necessary.

Water soluble polymers can be linked to the relaxin polypeptides of theinvention. The water soluble polymers may be linked via a non-naturallyencoded amino acid incorporated in the relaxin polypeptide or anyfunctional group or substituent of a non-naturally encoded or naturallyencoded amino acid, or any functional group or substituent added to anon-naturally encoded or naturally encoded amino acid. Alternatively,the water soluble polymers are linked to a relaxin polypeptideincorporating a non-naturally encoded amino acid via anaturally-occurring amino acid (including but not limited to, cysteine,lysine or the amine group of the N-terminal residue). In some cases, therelaxin polypeptides of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10 non-natural amino acids, wherein one or more non-naturally-encodedamino acid(s) are linked to water soluble polymer(s) (including but notlimited to, PEG and/or oligosaccharides). In some cases, the relaxinpolypeptides of the invention further comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more naturally-encoded amino acid(s) linked to water solublepolymers. In some cases, the relaxin polypeptides of the inventioncomprise one or more non-naturally encoded amino acid(s) linked to watersoluble polymers and one or more naturally-occurring amino acids linkedto water soluble polymers. In some embodiments, the water solublepolymers used in the present invention enhance the serum half-life ofthe relaxin polypeptide relative to the unconjugated form.

The number of water soluble polymers linked to a relaxin polypeptide(i.e., the extent of PEGylation or glycosylation) of the presentinvention can be adjusted to provide an altered (including but notlimited to, increased or decreased) pharmacologic, pharmacokinetic orpharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of relaxin is increased at least about 10,20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold,40-fold, 50-fold, or at least about 100-fold over an unmodifiedpolypeptide.

PEG Derivatives Containing a Strong Nucleophilic Group (i.e., Hydrazide,Hydrazine, Hydroxylamine or Semicarbazide)

In one embodiment of the present invention, a relaxin polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified with a PEG derivative that contains a terminal hydrazine,hydroxylamine, hydrazide or semicarbazide moiety that is linked directlyto the PEG backbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:RO—(CH2CH2O)n-O—(CH2)m-O—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:RO—(CH2CH2O)n-O—(CH2)m-X—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure:RO—(CH2CH2O)n-O—(CH2)m-NH—C(O)—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a relaxin polypeptide comprisinga carbonyl-containing amino acid is modified with a PEG derivative thatcontains a terminal hydroxylamine, hydrazide, hydrazine, orsemicarbazide moiety that is linked to the PEG backbone by means of anamide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)(CH2)m-O—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)(CH2)m-X—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is100-1,000 and X is optionally a carbonyl group (C═O) that can be presentor absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure:RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)(CH2)m-NH—C(O)—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a relaxin polypeptide comprisinga carbonyl-containing amino acid is modified with a branched PEGderivative that contains a terminal hydrazine, hydroxylamine, hydrazideor semicarbazide moiety, with each chain of the branched PEG having a MWranging from 10-40 kDa and, may be from 5-20 kDa.

In another embodiment of the invention, a relaxin polypeptide comprisinga non-naturally encoded amino acid is modified with a PEG derivativehaving a branched structure. For instance, in some embodiments, thehydrazine- or hydrazide-terminal PEG derivative will have the followingstructure:[RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)]2CH(CH2)m-X—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:[RO—(CH2CH2O)n-O—(CH2)2-C(O)—NH—CH2-CH2]2CH—X—(CH2)m-NH—C(O)—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:[RO—(CH2CH2O)n-O—(CH2)2-C(O)—NH—CH2-CH2]2CH—X—(CH2)m-O—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe relaxin polypeptide can modulate the binding of the relaxinpolypeptide to the relaxin polypeptide receptor. In some embodiments,the linkages are arranged such that the relaxin polypeptide binds therelaxin polypeptide receptor with a Kd of about 400 nM or lower, with aKd of 150 nM or lower, and in some cases with a Kd of 100 nM or lower,as measured by an equilibrium binding assay, such as that described inSpencer et al., J. Biol. Chem., 263:7862-7867 (1988).

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.: Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example. Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. Nos.5,219,564, 5,122,614, WO 90/13540, U.S. Pat. No. 5,281,698, and WO93/15189, and for conjugation between activated polymers and enzymesincluding but not limited to Coagulation Factor VIII (WO 94/15625),hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-52 (1985)). All references and patents citedare incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) of relaxinpolypeptides containing a non-naturally encoded amino acid, such asp-azido-L-phenylalanine, is carried out by any convenient method. Forexample, relaxin polypeptide is PEGylated with an alkyne-terminated mPEGderivative. Briefly, an excess of solid mPEG(5000)-O—CH2-C

CH is added, with stirring, to an aqueous solution ofp-azido-L-Phe-containing relaxin polypeptide at room temperature.Typically, the aqueous solution is buffered with a buffer having a pKanear the pH at which the reaction is to be carried out (generally aboutpH 4-10). Examples of suitable buffers for PEGylation at pH 7.5, forinstance, include, but are not limited to, HEPES, phosphate, borate,TRIS-HCl, EPPS, and TES. The pH is continuously monitored and adjustedif necessary. The reaction is typically allowed to continue for betweenabout 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated relaxin polypeptidevariants from free mPEG(5000)-O—CH2-C≡CH and any high-molecular weightcomplexes of the pegylated relaxin polypeptide which may form whenunblocked PEG is activated at both ends of the molecule, therebycrosslinking relaxin polypeptide variant molecules. The conditionsduring hydrophobic interaction chromatography are such that freemPEG(5000)-O—CH2-C≡CH flows through the column, while any crosslinkedPEGylated relaxin polypeptide variant complexes elute after the desiredforms, which contain one relaxin polypeptide variant molecule conjugatedto one or more PEG groups. Suitable conditions vary depending on therelative sizes of the cross-linked complexes versus the desiredconjugates and are readily determined by those of ordinary skill in theart. The eluent containing the desired conjugates is concentrated byultrafiltration and desalted by diafiltration.

If necessary, the PEGylated relaxin polypeptide obtained from thehydrophobic chromatography can be purified further by one or moreprocedures known to those of ordinary skill in the art including, butare not limited to, affinity chromatography; anion- or cation-exchangechromatography (using, including but not limited to, DEAE SEPHAROSE);chromatography on silica; reverse phase HPLC; gel filtration (using,including but not limited to, SEPHADEX G-75); hydrophobic interactionchromatography; size-exclusion chromatography, metal-chelatechromatography; ultrafiltration/diafiltration; ethanol precipitation;ammonium sulfate precipitation; chromatofocusing; displacementchromatography; electrophoretic procedures (including but not limited topreparative isoelectric focusing), differential solubility (includingbut not limited to ammonium sulfate precipitation), or extraction.Apparent molecular weight may be estimated by GPC by comparison toglobular protein standards (Preneta, A Z in PROTEIN PURIFICATIONMETHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989,293-306). The purity of the relaxin-PEG conjugate can be assessed byproteolytic degradation (including but not limited to, trypsin cleavage)followed by mass spectrometry analysis. Pepinsky R B., et al., J.Pharmcol. & Exp. Ther. 297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid of a relaxin polypeptideof the invention can be further derivatized or substituted withoutlimitation.

Azide-containing PEG derivatives

In another embodiment of the invention, a relaxin polypeptide ismodified with a PEG derivative that contains an azide moiety that w %ill react with an alkyne moiety present on the side chain of thenon-naturally encoded amino acid. In general, the PEG derivatives willhave an average molecular weight ranging from 1-100 kDa and, in someembodiments, from 1040 kDa.

In some embodiments, the azide-terminal PEG derivative will have thestructure:RO—(CH2CH2O)n-O—(CH2)m-N3

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative will have thestructure:RO—(CH2CH2O)n-O—(CH2)m-NH—C(O)—(CH2)p-N3

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40kDa).

In another embodiment of the invention, a relaxin polypeptide comprisinga alkyne-containing amino acid is modified with a branched PEGderivative that contains a terminal azide moiety, with each chain of thebranched PEG having a MW ranging from 10-40 kDa and may be from 5-20kDa. For instance, in some embodiments, the azide-terminal PEGderivative will have the following structure:[RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)]2CH(CH2)m-X—(CH2)pN3

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), in each case that can be present or absent.

Alkyne-Containing PEG Derivatives

In another embodiment of the invention, a relaxin polypeptide ismodified with a PEG derivative that contains an alkyne moiety that willreact with an azide moiety present on the side chain of thenon-naturally encoded amino acid.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:RO—(CH2CH2O)n-O—(CH2)m-C

CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, a relaxin polypeptide comprisingan alkyne-containing non-naturally encoded amino acid is modified with aPEG derivative that contains a terminal azide or terminal alkyne moietythat is linked to the PEG backbone by means of an amide linkage.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:RO—(CH2CH2O)n-O—(CH2)m-NH—C(O)—(CH2)p-C

CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000.

In another embodiment of the invention, a relaxin polypeptide comprisingan azide-containing amino acid is modified with a branched PEGderivative that contains a terminal alkyne moiety, with each chain ofthe branched PEG having a MW ranging from 10-40 kDa and may be from 5-20kDa. For instance, in some embodiments, the alkyne-terminal PEGderivative will have the following structure:[RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)]2CH(CH2)m-X—(CH2)pC

CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), or not present.

Phosphine-Containing PEG Derivatives

In another embodiment of the invention, a relaxin polypeptide ismodified with a PEG derivative that contains an activated functionalgroup (including but not limited to, ester, carbonate) furthercomprising an aryl phosphine group that will react with an azide moietypresent on the side chain of the non-naturally encoded amino acid. Ingeneral, the PEG derivatives will have an average molecular weightranging from 1-100 kDa and, in some embodiments, from 10-40 kDa.

In some embodiments the PEG derivative will have the structure:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

In some embodiments, the PEG derivative will have the structure:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)2NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,I-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Other PEG Derivatives and General PEGylation Techniques

Other exemplary PEG molecules that may be linked to relaxinpolypeptides, as well as PEGylation methods include, but are not limitedto, those described in, e.g., U.S. Patent Publication No. 2004/0001838;2002/0052009; 2003/0162949; 2004/0013637; 2003/0228274; 2003/0220447;2003/0158333; 2003/0143596; 2003/0114647; 2003/0105275; 2003/0105224;2003/0023023; 2002/0156047; 2002/0099133; 2002/0086939; 2002/0082345;2002/0072573; 2002/0052430; 2002/0040076; 2002/0037949; 2002/0002250;2001/0056171; 2001/0044526; 2001/0021763; U.S. Pat. Nos. 6,646,110;5,824,778; 5,476,653; 5,219,564; 5,629,384; 5,736,625; 4,902,502;5,281,698; 5,122,614; 5,473,034; 5,516,673; 5,382,657; 6,552,167;6,610,281; 6,515,100; 6,461,603; 6,436,386; 6,214,966; 5,990,237;5,900,461; 5,739,208; 5,672,662; 5,446,090; 5,808,096; 5,612,460;5,324,844; 5,252,714; 6,420,339; 6,201,072; 6,451,346; 6,306,821;5,559,213; 5,747,646; 5,834,594; 5,849,860; 5,980,948; 6,004,573;6,129,912; WO 97/32607, EP 229,108, EP 402,378, WO 92/16555, WO94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO97/03106, WO 96/21469, WO 95/13312, EP 921 131, WO 98/05363, EP 809 996,WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400 472, EP 183 503and EP 154 316, which are incorporated by reference herein. Any of thePEG molecules described herein may be used in any form, including butnot limited to, single chain, branched chain, multiarm chain, singlefunctional, bi-functional, multi-functional, or any combination thereof.

Additional polymer and PEG derivatives including but not limited to,hydroxylamine (aminooxy) PEG derivatives, are described in the followingpatent applications which are all incorporated by reference in theirentirety herein: U.S. Patent Publication No. 2006/0194256, U.S. PatentPublication No. 2006/0217532, U.S. Patent Publication No. 2006/0217289,U.S. Provisional Patent No. 60/755,338; U.S. Provisional Patent No.60/755,711; U.S. Provisional Patent No. 60/755,018: International PatentApplication No. PCT/US06/49397; WO 2006/069246; U.S. Provisional PatentNo. 60/743,041; U.S. Provisional Patent No. 60/743,040; InternationalPatent Application No. PCT/US06/47822; U.S. Provisional Patent No.60/882,819; U.S. Provisional Patent No. 60/882,500; and U.S. ProvisionalPatent No. 60/870,594.

Heterologous Fc Fusion Proteins

The relaxin compounds described above may be fused directly or via apeptide linker to the Fc portion of an immunoglobulin. Immunoglobulinsare molecules containing polypeptide chains held together by disulfidebonds, typically having two light chains and two heavy chains. In eachchain, one domain (V) has a variable amino acid sequence depending onthe antibody specificity of the molecule. The other domains (C) have arather constant sequence common to molecules of the same class.

As used herein, the Fc portion of an immunoglobulin has the meaningcommonly given to the term in the field of immunology. Specifically,this term refers to an antibody fragment which is obtained by removingthe two antigen binding regions (the Fab fragments) from the antibody.One way to remove the Fab fragments is to digest the immunoglobulin withpapain protease. Thus, the Fc portion is formed from approximately equalsized fragments of the constant region from both heavy chains, whichassociate through non-covalent interactions and disulfide bonds. The Fcportion can include the hinge regions and extend through the CH2 and CH3domains to the C-terminus of the antibody. Representative hinge regionsfor human and mouse immunoglobulins can be found in AntibodyEngineering, A Practical Guide, Borrebaeck, C. A. K., ed., W. H. Freemanand Co., 1992, the teachings of which are herein incorporated byreference. The Fc portion can further include one or more glycosylationsites. The amino acid sequences of numerous representative Fc proteinscontaining a hinge region, CH2 and CH3 domains, and one N-glycosylationsite are well known in the art.

There are five types of human immunoglobulin Fc regions with differenteffector functions and pharmacokinetic properties: IgG, IgA, IgM, IgD,and IgE. IgG is the most abundant immunoglobulin in serum. IgG also hasthe longest half-life in serum of any immunoglobulin (23 days). Unlikeother immunoglobulins, IgG is efficiently recirculated following bindingto an Fc receptor. There are four IgG subclasses G1, G2, G3, and G4,each of which has different effector functions. G1, G2, and G3 can bindC1q and fix complement while G4 cannot. Even though G3 is able to bindC1q more efficiently than G1, G1 is more effective at mediatingcomplement-directed cell lysis. G2 fixes complement very inefficiently.The C1q binding site in IgG is located at the carboxy terminal region ofthe CH2 domain.

All IgG subclasses are capable of binding to Fc receptors (CD16, CD32,CD64) with G1 and G3 being more effective than G2 and G4. The Fcreceptor binding region of IgG is formed by residues located in both thehinge and the carboxy terminal regions of the CH2 domain.

IgA can exist both in a monomeric and dimeric form held together by aJ-chain. IgA is the second most abundant Ig in serum, but it has ahalf-life of only 6 days. IgA has three effector functions. It binds toan IgA specific receptor on macrophages and eosinophils, which drivesphagocytosis and degranulation, respectively. It can also fix complementvia an unknown alternative pathway.

IgM is expressed as either a pentamer or a hexamer, both of which areheld together by a J-chain. IgM has a serum half-life of 5 days. Itbinds weakly to C1q via a binding site located in its CH3 domain. IgDhas a half-life of 3 days in serum. It is unclear what effectorfunctions are attributable to this Ig. IgE is a monomeric Ig and has aserum half-life of 2.5 days. IgE binds to two Fc receptors which drivesdegranulation and results in the release of proinflammatory agents.

Depending on the desired in vivo effect, the heterologous fusionproteins of the present invention may contain any of the isotypesdescribed above or may contain mutated Fc regions wherein the complementand/or Fc receptor binding functions have been altered. Thus, theheterologous fusion proteins of the present invention may contain theentire Fc portion of an immunoglobulin, fragments of the Fc portion ofan immunoglobulin, or analogs thereof fused to an interferon betacompound.

The fusion proteins of the present invention can consist of single chainproteins or as multi-chain polypeptides. Two or more Fc fusion proteinscan be produced such that they interact through disulfide bonds thatnaturally form between Fc regions. These multimers can be homogeneouswith respect to the interferon beta compound or they may containdifferent interferon beta compounds fused at the N-terminus of the Fcportion of the fusion protein.

Regardless of the final structure of the fusion protein, the Fc orFc-like region may serve to prolong the in vivo plasma half-life of theinterferon beta compound fused at the N-terminus. Also, the interferonbeta component of a fusion protein compound should retain at least onebiological activity of interferon beta. An increase in therapeutic orcirculating half-life can be demonstrated using the method describedherein or known in the art, wherein the half-life of the fusion proteinis compared to the half-life of the interferon beta compound alone.Biological activity can be determined by in vitro and in vivo methodsknown in the art.

Since the Fc region of IgG produced by proteolysis has the same in vivohalf-life as the intact IgG molecule and Fab fragments are rapidlydegraded, it is believed that the relevant sequence for prolonginghalf-life reside in the CH2 and/or CH3 domains. Further, it has beenshown in the literature that the catabolic rates of IgG variants that donot bind the high-affinity Fc receptor or C1q are indistinguishable fromthe rate of clearance of the parent wild-type antibody, indicating thatthe catabolic site is distinct from the sites involved in Fc receptor orC1q binding. [Wamrzynczak et al., (1992) Molecular Immunology 29:221].Site-directed mutagenesis studies using a murine IgG1 Fc regionsuggested that the site of the IgG1 Fc region that controls thecatabolic rate is located at the CH2-CH3 domain interface. Fc regionscan be modified at the catabolic site to optimize the half-life of thefusion proteins. The Fc region used for the fusion proteins of thepresent invention may be derived from an IgG1 or an IgG4 Fc region, andmay contain both the CH2 and CH3 regions including the hinge region.

Heterologous Albumin Fusion Proteins

Relaxin described herein may be fused directly or via a peptide linker,water soluble polymer, or prodrug linker to albumin or an analog,fragment, or derivative thereof. Generally, the albumin proteins thatare part of the fusion proteins of the present invention may be derivedfrom albumin cloned from any species, including human. Human serumalbumin (HSA) consists of a single non-glycosylated polypeptide chain of585 amino acids with a formula molecular weight of 66,500. The aminoacid sequence of human HSA is known [See Meloun, et al. (1975) FEBSLetters 58:136; Behrens, et al. (1975) Fed. Proc. 34:591; Lawn, et al.(1981) Nucleic Acids Research 9:6102-6114; Minghetti, et al. (1986) J.Biol. Chem. 261:6747, each of which are incorporated by referenceherein]. A variety of polymorphic variants as well as analogs andfragments of albumin have been described. [See Weitkamp, et al., (1973)Ann. Hum. Genet. 37:219]. For example, in EP 322,094, various shorterforms of HSA. Some of these fragments of HSA are disclosed, includingHSA(1-373), HSA(1-388), HSA(1-389), HSA(I-369), and HSA(1-419) andfragments between 1-369 and 1-419. EP 399,666 discloses albuminfragments that include HSA(1-177) and HSA(1-200) and fragments betweenHSA(1-177) and HSA(1-200).

It is understood that the heterologous fusion proteins of the presentinvention include relaxin compounds that are coupled to any albuminprotein including fragments, analogs, and derivatives wherein suchfusion protein is biologically active and has a longer plasma half-lifethan the relaxin compound alone. Thus, the albumin portion of the fusionprotein need not necessarily have a plasma half-life equal to that ofnative human albumin. Fragments, analogs, and derivatives are known orcan be generated that have longer half-lives or have half-livesintermediate to that of native human albumin and the relaxin compound ofinterest.

The heterologous fusion proteins of the present invention encompassproteins having conservative amino acid substitutions in the relaxincompound and/or the Fc or albumin portion of the fusion protein. A“conservative substitution” is the replacement of an amino acid withanother amino acid that has the same net electronic charge andapproximately the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have approximately the samesize when the total number carbon and heteroatoms in their side chainsdiffers by no more than about four. They have approximately the sameshape when the number of branches in their side chains differs by nomore than one. Amino acids with phenyl or substituted phenyl groups intheir side chains are considered to have about the same size and shape.Except as otherwise specifically provided herein, conservativesubstitutions are preferably made with naturally occurring amino acids.

Wild-type albumin and immunoglobulin proteins can be obtained from avariety of sources. For example, these proteins can be obtained from acDNA library prepared from tissue or cells which express the mRNA ofinterest at a detectable level. Libraries can be screened with probesdesigned using the published DNA or protein sequence for the particularprotein of interest. For example, immunoglobulin light or heavy chainconstant regions are described in Adams, et al. (1980) Biochemistry19:2711-2719; Goughet, et al. (1980) Biochemistry 19:2702-2710; Dolby,et al. (1980) Proc. Natl. Acad. Sci. USA 77:6027-6031; Rice et al.(1982) Proc. Natl. Acad. Sci. USA 79:7862-7862: Falkner, et al. (1982)Nature 298:286-288; and Morrison, et al. (1984) Ann. Rev. Immunol.2:239-256. Some references disclosing albumin protein and DNA sequencesinclude Meloun, et al. (1975) FEBS Letters 58:136; Behrens, et al.(1975) Fed. Proc. 34:591; Lawn, et al. (1981) Nucleic Acids Research9:6102-6114; and Minghetti, et al. (1986) J. Biol. Chem. 261:6747.

Characterization of the Heterologous Fusion Proteins of the PresentInvention

Numerous methods exist to characterize the fusion proteins of thepresent invention. Some of these methods include, but are not limitedto: SDS-PAGE coupled with protein staining methods or immunoblottingusing anti-IgG or anti-HSA antibodies. Other methods include matrixassisted laser desorption/ionization-mass spectrometry (MALDI-MS),liquid chromatography/mass spectrometry, isoelectric focusing,analytical anion exchange, chromatofocusing, and circular dichroism, forexample.

Enhancing Affinity for Serum Albumin

Various molecules can also be fused to the relaxin polypeptides of theinvention to modulate the half-life of relaxin polypeptides in serum. Insome embodiments, molecules are linked or fused to relaxin polypeptidesof the invention to enhance affinity for endogenous serum albumin in ananimal.

For example, in some cases, a recombinant fusion of a relaxinpolypeptide and an albumin binding sequence is made. Exemplary albuminbinding sequences include, but are not limited to, the albumin bindingdomain from streptococcal protein G (see. e.g., Makrides et al., J.Pharmacol. Exp. Ther. 277:534-542 (1996) and Sjolander et al., J,Immunol. Methods 201:115-123 (1997)), or albumin-binding peptides suchas those described in, e.g., Dennis, et al., J. Biol. Chem.277:35035-35043 (2002).

In other embodiments, the relaxin polypeptides of the present inventionare acylated with fatty acids. In some cases, the fatty acids promotebinding to serum albumin. See, e.g., Kurtzhals, et al., Biochem. J.312:725-731 (1995).

In other embodiments, the relaxin polypeptides of the invention arefused directly with serum albumin (including but not limited to, humanserum albumin). Those of skill in the art will recognize that a widevariety of other molecules can also be linked to relaxin in the presentinvention to modulate binding to serum albumin or other serumcomponents.

Glycosylation of Relaxin Polypeptides

The invention includes relaxin polypeptides incorporating one or morenon-naturally encoded amino acids bearing saccharide residues. Thesaccharide residues may be either natural (including but not limited to,N-acetylglucosamine) or non-natural (including but not limited to,3-fluorogalactose). The saccharides may be linked to the non-naturallyencoded amino acids either by an N- or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to relaxin polypeptides either in vivo or in vitro. In someembodiments of the invention, a relaxin polypeptide comprising acarbonyl-containing non-naturally encoded amino acid is modified with asaccharide derivatized with an aminooxy group to generate thecorresponding glycosylated polypeptide linked via an oxime linkage. Onceattached to the non-naturally encoded amino acid, the saccharide may befurther elaborated by treatment with glycosyltransferases and otherenzymes to generate an oligosaccharide bound to the relaxin polypeptide.See, e.g., H. Liu, et al. J. Am. Chem. Soc. 125: 1702-1703 (2003).

In some embodiments of the invention, a relaxin polypeptide comprising acarbonyl-containing non-naturally encoded amino acid is modifieddirectly with a glycan with defined structure prepared as an aminooxyderivative. One of ordinary skill in the art will recognize that otherfunctionalities, including azide, alkyne, hydrazide, hydrazine, andsemicarbazide, can be used to link the saccharide to the non-naturallyencoded amino acid.

In some embodiments of the invention, a relaxin polypeptide comprisingan azide or alkynyl-containing non-naturally encoded amino acid can thenbe modified by, including but not limited to, a Huisgen [3+2]cycloaddition reaction with, including but not limited to, alkynyl orazide derivatives, respectively. This method allows for proteins to bemodified with extremely high selectivity.

Relaxin Dimers and Multimers

The present invention also provides for relaxin and relaxin analogcombinations such as homodimers, heterodimers, homomultimers, orheteromultimers (i.e., trimers, tetramers, etc.) where relaxincontaining one or more non-naturally encoded amino acids is bound toanother relaxin or relaxin variant thereof or any other polypeptide thatis not relaxin or relaxin variant thereof, either directly to thepolypeptide backbone or via a linker. Due to its increased molecularweight compared to monomers, the relaxin dimer or multimer conjugatesmay exhibit new or desirable properties, including but not limited todifferent pharmacological, pharmacokinetic, pharmacodynamic, modulatedtherapeutic half-life, or modulated plasma half-life relative to themonomeric relaxin. For examples of monomeric relaxin analogs see, forexample, Balschmidt, P., et al., U.S. Pat. No. 5,164,366, issued Nov.17, 1992; Brange, J., et al., U.S. Pat. No. 5,618,913, issued Apr. 8,1997; Chance, R. E., et al., U.S. Pat. No. 5,514,646, issued May 7,1996; and Ertl, J., et al., EPO publication number 885,961, Dec. 23,1998. Some embodiments of the present invention provide monomericrelaxin analogs containing one or more non-naturally encoded amino acidresidues and in some embodiments these include monomeric relaxin analogswherein position B28 is Asp, Lys, Ile, Leu, Val or Ala and the aminoacid residue at position B29 is Lys or Pro, monomeric relaxin analogwith Lys(B28)Pro(B29)-human relaxin; monomeric relaxin analogAsp(B28)-human relaxin; and monomeric relaxin analogLys(B3)Ile(B28)-human relaxin. In some embodiments, relaxin dimers ofthe invention will modulate signal transduction of the relaxin receptor.In other embodiments, the relaxin dimers or multimers of the presentinvention will act as a relaxin receptor antagonist, agonist, ormodulator.

In some embodiments, one or more of the relaxin molecules present in arelaxin containing dimer or multimer comprises a non-naturally encodedamino acid linked to a water soluble polymer.

In some embodiments, the relaxin polypeptides are linked directly,including but not limited to, via an Asn-Lys amide linkage or Cys-Cysdisulfide linkage. In some embodiments, the relaxin polypeptides, and/orthe linked non-relaxin molecule, will comprise different non-naturallyencoded amino acids to facilitate dimerization, including but notlimited to, an alkyne in one non-naturally encoded amino acid of a firstrelaxin polypeptide and an azide in a second non-naturally encoded aminoacid of a second molecule will be conjugated via a Huisgen [3+2]cycloaddition. Alternatively, relaxin, and/or the linked non-relaxinmolecule comprising a ketone-containing non-naturally encoded amino acidcan be conjugated to a second polypeptide comprising ahydroxylamine-containing non-naturally encoded amino acid and thepolypeptides are reacted via formation of the corresponding oxime.

Alternatively, the two relaxin polypeptides, and/or the linkednon-relaxin molecule, are linked via a linker. Any hetero- orhomo-bifunctional linker can be used to link the two molecules, and/orthe linked non-relaxin molecules, which can have the same or differentprimary sequence. In some cases, the linker used to tether the relaxin,and/or the linked non-relaxin molecules together can be a bifunctionalPEG reagent. The linker may have a wide range of molecular weight ormolecular length. Larger or smaller molecular weight linkers may be usedto provide a desired spatial relationship or conformation betweenrelaxin and the linked entity or between relaxin and its receptor, orbetween the linked entity and its binding partner, if any. Linkershaving longer or shorter molecular length may also be used to provide adesired space or flexibility between relaxin and the linked entity, orbetween the linked entity and its binding partner, if any.

In some embodiments, the invention provides water-soluble bifunctionallinkers that have a dumbbell structure that includes: a) an azide, analkyne, a hydrazine, a hydrazide, a hydroxylamine, or acarbonyl-containing moiety on at least a first end of a polymerbackbone; and b) at least a second functional group on a second end ofthe polymer backbone. The second functional group can be the same ordifferent as the first functional group. The second functional group, insome embodiments, is not reactive with the first functional group. Theinvention provides, in some embodiments, water-soluble compounds thatcomprise at least one arm of a branched molecular structure. Forexample, the branched molecular structure can be dendritic.

In some embodiments, the invention provides multimers comprising one ormore relaxin polypeptide, formed by reactions with water solubleactivated polymers that have the structure:R—(CH2CH2O)n-O—(CH2)m-X

wherein n is from about 5 to 3,000, m is 2-10, X can be an azide, analkyne, a hydrazine, a hydrazide, an aminooxy group, a hydroxylamine, anacetyl, or carbonyl-containing moiety, and R is a capping group, afunctional group, or a leaving group that can be the same or differentas X. R can be, for example, a functional group selected from the groupconsisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester.N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetanide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.

Measurement of Relaxin Polypeptide Activity and Affinity of RelaxinPolypeptide for the Relaxin Receptor

Relaxin polypeptide activity can be determined using standard or knownin vitro or in vivo assays. Relaxin polypeptides may be analyzed forbiological activity by suitable methods known in the art. Such assaysinclude, but are not limited to, receptor binding assays,anti-proliferative assays (Samuel et al., Endocrinology145(9):4125-4133), and cell signaling assays including but not limitedto cellular cAMP induction.

With respect to diseases such as heart failure and fibrotic disease,activity of a relaxin (such as RLX-A-AQ1-20KPEG) may also be determinedusing one or more in vivo assays. Said assays typically involveadministration of a relaxin in an animal model of said disease anddetermining the effect on disease progression, severity, or otherindicia of efficacious treatment. Such assays may be used to determineefficacious dosages and treatment regimens in the model system, andbased thereon predict a dosing regimen for clinical use, e.g., for humanpatients. Exemplary HF assays that may be utilized include: 1) the mouseleft anterior descending (LAD) coronary ligation model which mimics thecardiac changes of patients suffering a myocardial infarction andprogression to HF (Samuel et al., Lab. Investigation 91:675-690, 2011);2) limited AngII-infusion model in which minimal concentrations AngIIare utilized to induce cardiac fibrosis (Xu et al., J. Cardiovasc.Pharmacol. 51:62-70, 2008); 3) Dahl-Salt sensitive rat model which ischaracterized by hypertension, renal impairment, and blood volumeoverload (Sakata et al., Circulation 109:2143-2149, 2004); 4)Aged-Spontaneously Hypertensive Rat (SHR) model of cardiac and renalfibrosis which was previously utilized to demonstrate WT-RLX efficacy(Lekgabe et al., Hypertension 46:412-418, 2005); and 5) rat thoracicaortic constriction model of pressure overload (Kuster et al.,Circulation 111:420-427, 2005). In addition to these models, activity ofrelaxins such as RLX-A-AQ1-20KPEG may also be determined in a dog model,such as in normal and tachypacing-induced HF dogs. Additionally, theseassays may be performed to determine whether a relaxin such asRLX-A-AQ1-20KPEG is efficacious when given not only in preventativemode, but also in therapeutic mode. Further, relaxins such asRLX-A-AQ1-20KPEG may be tested in models of fibrosis including renal(Yoshida et al., Nephrol. Dialysis Transplant 27: 2190-2197, 2012), lung(Huang et al., Am. J. Pathol. 179:2751-2765, 2011), and liver fibrosis(Williams et al., Gut 49:577-583, 2001). For example, efficacy ofRLX-A-AQ1-20KPEG may be compared to efficacy of wild-type relaxin (e.g.,wild-type human relaxin).

Average quantities of relaxin, relaxin polypeptides, and/or relaxinanalogues of the present invention may vary and in particular should bebased upon the recommendations and prescription of a qualifiedphysician. The exact amount of relaxin, relaxin polypeptides, and/orrelaxin analogues of the present invention is a matter of preferencesubject to such factors as the exact type and/or severity of thecondition being treated, the condition of the patient being treated, aswell as the other ingredients in the composition. The invention alsoprovides for administration of a therapeutically effective amount ofanother active agent. The amount to be given may be readily determinedby one of ordinary skill in the art based upon therapy with relaxin,available relaxin therapies, and/or other relaxin analogues.

Pharmaceutical compositions of the invention may be manufactured in aconventional manner.

The invention will now be described in more detail with respect to thefollowing, specific, non-limiting examples.

EXAMPLES Example 1

This example describes one of the many potential sets of criteria forthe selection of sites of incorporation of non-naturally encoded aminoacids into relaxin.

FIGS. 1-4 show the structure and the sequence of relaxin and the tablebelow includes sequences with the A chain, B chain, relaxin andprorelaxin. Relaxin polypeptides were generated by substituting anaturally encoded amino acid with a non-naturally encoded amino acid.Each polypeptide had one of the amino acids substituted withpara-acetylphenylalanine (pAcF or pAF). The polypeptides generatedlacked the leader sequence and were A/B chain relaxin polypeptides (SEQID NO. 1-3). Each of the polypeptides generated had a non-naturallyencoded amino acid substitution at one of the following positions 1, 5,18, 13, 2 of SEQ ID NO: 4 or in those positions of the A chain of any ofthe known relaxin sequences or 5, 7, 18, 28 of SEQ ID NO: 5 or 6 inthose same positions of the B chain of any of the known relaxinsequences. FIG. 2 shows the structure of human relaxin that was labeledusing the PyMOL software (DeLano Scientific; Palo Alto, CA) and someamino acids corresponding to those substituted withpara-acetylphenylalanine in relaxin polypeptides of the invention.

Another set of criteria for the selection of preferred sites ofincorporation of non-naturally encoded amino acids includes using andcomparing crystal structures from the Protein Data Bank, or other databanks, are used to model the structure of relaxin and residues areidentified that 1) would not interfere with binding to their receptor,and 2) would not be present in the interior of the protein. In someembodiments, one or more non-naturally encoded amino acids areincorporated at, but not limited to, one or more of the followingpositions of relaxin: 1, 5, 18, 13, 2 of SEQ ID NO: 4 or in thosepositions of the A chain of any of the known relaxin sequences or 5, 7,18, 28 of SEQ ID NO: 5 or 6 in those same positions of the B chain ofany of the known relaxin sequences.

The following criteria were used to evaluate each position of relaxinand relaxin analogs for the introduction of a non-naturally encodedamino acid: the residue (a) should not interfere with binding of thereceptor based on structural analysis, b) should not be affected byalanine or homolog scanning mutagenesis (c) should be surface exposedand exhibit minimal van der Waals or hydrogen bonding interactions withsurrounding residues, (d) should be either deleted or variable inrelaxin variants, (e) would result in conservative changes uponsubstitution with a non-naturally encoded amino acid and (f) could befound in either highly flexible regions or structurally rigid regions.In addition, further calculations can be performed on the relaxinmolecule, utilizing the Cx program (Pintar et al. (2002) Bioinformatics,18, pp 980) to evaluate the extent of protrusion for each protein atom.

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in the A chain ofrelaxin: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25(i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 4 or thecorresponding amino acids in SEQ ID NOs: 1-3). In some embodiments, oneor more non-naturally encoded amino acids are incorporated in one ormore of the following positions in the B chain of relaxin: beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 (i.e., at the carboxyl terminus of the protein) (SEQ ID NO: 5 or 6 orthe corresponding amino acids in SEQ ID NOs: 1-3). In some embodiments,one or more non-naturally encoded amino acids are incorporated in one ormore of the following positions in relaxin: 1, 5, 31, 2, 13, 29, 18, 52(SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs: 2 and 3).In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in relaxin: 5,31, 2, 13, 29, 18, 52 (SEQ ID NO: 1 or the corresponding amino acids inSEQ ID NOs: 2 and 3). In some embodiments, one or more non-naturallyencoded amino acids are incorporated in one or more of the followingpositions in relaxin: 1, 5, 31, 2, 13, 29 (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 2 and 3). In some embodiments,one or more non-naturally encoded amino acids are incorporated in one ormore of the following positions in relaxin: 5, 31, 2, 13, 29 (SEQ ID NO:1 or the corresponding amino acids in SEQ ID NOs: 2 and 3). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in relaxin: 1, 5,31, 2, 13 (SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs:2 and 3). In some embodiments, one or more non-naturally encoded aminoacids are incorporated in one or more of the following positions inrelaxin: 5, 31, 2, 13 (SEQ ID NO: 1 or the corresponding amino acids inSEQ ID NOs: 2 and 3).

Example 2

This example details cloning and expression of a relaxin polypeptideincluding a non-naturally encoded amino acid in E. coli.

Methods for cloning relaxin are known to those of ordinary skill in theart. Polypeptide and polynucleotide sequences for relaxin and cloning ofrelaxin into host cells are detailed in U.S. Pat. Nos. 4,758,516;5,166,191, 5,179,195, 5,945,402; and 5,759,807, all of which patents areherein incorporated by reference.

cDNA encoding relaxin is shown as SEQ ID NOs: 12 and the maturepolypeptide amino acid sequence is shown as SEQ ID NO: 1.

TABLE 1 Relaxin Sequences Cited SEQ ID NO: Sequence Name Sequence 1Relaxin amino acid QLYSALANKCCHVGCTKRSLARFC sequenceDSWMEEVIKLCGRELVRAQIAICGMSTWS 2 Relaxin amino acidQLYSALANKCCHVGCTKRSLARFC sequence B1 Ala ASWMEEVIKLCGRELVRAQIAICGMSTWS 3Pro-relaxin amino acid DSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQVGQVEsequence LGGPGAGSLQPLALEGSLQKRQLYSALANKCCHVGCTKRSL ARFC 4Relaxin A chain, amino QLYSALANKCCHVGCTKRSLARFC acid sequence 5Relaxin B chain, amino DSWMEEVIKLCGRELVRAQIAICGMSTWS acid sequence 6Relaxin B chain, amino ASWMEEVIKLCGRELVRAQIAICGMSTWSacid sequence with B1 Ala 7 C peptide amino acidRREAEDLQVGWELGGGPGAGSLQPLALEGSLQKR sequence 8 Relaxin leader amino acidMKKINIAFLLKR sequence 9 Insulin leader amino acid MIEGGR sequence 10Relaxin A chain, nucleic caactctacagtgcattggctaataaatgttgccatgttggttgacid sequence taccaaaagatctcttgctagattttgc 11 Relaxin B chain, nucleicgactcatggatggaggaagttattaaattatgcggccgcgaattagttcgcgcgc acid sequenceagattgccatttgcggcatgagcacctggagc 12 Relaxin, A and B chains,caactctacagtgcattggctaataaatgttgccatgttggttgtaccaaaanucleic acid sequencegatctcttgctagattttgcgactcatggatggaggaagttattaaattatgcggccgcgaattagttcgcgcgcagattgccatttgcggcatgagcacctggagc 13Relaxin leader nucleic atgaaaaagaatatcgcatttcttcttaaacgg acid sequence14 Insulin leader nucleic  atgattgaaggtggtcgt acid sequence 15Example of a relaxin MIEGGRDSWMEEVIKLCGRELVRAQIAICGMSTWSRREAEDLQexpression construct VGQVELGGGPGAGSLQPLALEGSLQKRQLYSALANKCCHVGCamino acid sequence TKRSLARFC 35 Relaxin A chain, aminoXLYSALANKCCHVGCTKRSLARFC acid sequence with a non-naturally encoded amino acid at position 1 (X is pAcF)

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O—RS) is used toexpress relaxin or relaxin analogs containing a non-naturally encodedamino acid. The O—RS preferentially aminoacylates the O-tRNA with anon-naturally encoded amino acid. In turn the translation system insertsthe non-naturally encoded amino acid into the relaxin or relaxin analog,in response to an encoded selector codon. Suitable O—RS and O-tRNAsequences are described in WO 2006/068802 entitled “Compositions ofAminoacyl-tRNA Synthetase and Uses Thereof” (E9; SEQ ID NO: 16) and WO2007/021297 entitled “Compositions of” tRNA and Uses Thereof (F13; SEQID NO: 17), which are incorporated by reference in their entiretyherein.

TABLE 2 Sequences Cited SEQ ID NO: 18 M. jannaschii mtRNA_(CUA) ^(Tyr)tRNA SEQ ID NO: 19 HLAD03; an optimized amber supressor tRNA tRNA SEQ IDNO: 20 HL325A; an optimized AGGA frameshift tRNA supressor tRNA SEQ IDNO: 21 Aminoacyl tRNA synthetase for the RS incorporation ofp-azido-L-phenylalanine p-Az-PheRS(6) SEQ ID NO: 22 Aminoacyl tRNAsynthetase for the RS incorporation of p-benzoyl-L-phenylalaninep-BpaRS(1) SEQ ID NO: 23 Aminoacyl tRNA synthetase for the RSincorporation of propargyl-phenylalanine Propargyl-PheRS SEQ ID NO: 24Aminoacyl tRNA synthetase for the RS incorporation ofpropargyl-phenylalanine Propargyl-PheRS SEQ ID NO: 25 Aminoacyl tRNAsynthetase for the RS incorporation of propargyl-phenylalaninePropargyl-PheRS SEQ ID NO: 26 Aminoacyl tRNA synthetase for the RSincorporation of p-azido-phenylalanine p-Az-PheRS(1) SEQ ID NO: 27Aminoacyl tRNA synthetase for the RS incorporation ofp-azido-phenylalanine p-Az-PheRS(3) SEQ ID NO: 28 Aminoacyl tRNAsynthetase for the RS incorporation of p-azido-phenylalaninep-Az-PheRS(4) SEQ ID NO: 29 Aminoacyl tRNA synthetase for the RSincorporation of p-azido-phenylalanine p-Az-PheRS(2) SEQ ID NO: 30Aminoacyl tRNA synthetase for the RS incorporation ofp-acetyl-phenylalanine (LW1) SEQ ID NO: 31 Aminoacyl tRNA synthetase forthe RS incorporation of p-acetyl-phenylalanine (LW5) SEQ ID NO: 32Aminoacyl tRNA synthetase for the RS incorporation ofp-acetyl-phenylalanine (LW6) SEQ ID NO: 33 Aminoacyl tRNA synthetase forthe RS incorporation of p-azido-phenylalanine (AzPheRS-5) SEQ ID NO: 34Aminoacyl tRNA synthetase for the RS incorporation ofp-azido-phenylalanine (AzPheRS-6)

The transformation of E. coli with plasmids containing the modifiedrelaxin or relaxin analog gene and the orthogonal aminoacyl tRNAsynthetase/tRNA pair (specific for the desired non-naturally encodedamino acid) allows the site-specific incorporation of non-naturallyencoded amino acid into the relaxin polypeptide.

Wild type mature relaxin is amplified by PCR from a cDNA synthesisreaction using standard protocols and cloned into pET30 (NcoI-BamHI).Prior to or alternatively following sequence confirmation, relaxinincluding an N-terminal HHHHHHSGG (SEQ ID NO: 36) sequence is subclonedinto a suppression vector containing an amber suppressor tyrosyltRNATyr/CUA from Methanococcus jannaschii (Mj tRNATyr/CUA) underconstitutive control of a synthetic promoter derived from the E. colilipoprotein promoter sequence (Miller, J. H., Gene, 1986), as well aswell as the orthogonal tyrosyl-tRNA-synthetase (MjTyrRS) under controlof the E. coli GlnRS promoter. Expression of relaxin is under control ofthe T7 promoter. Amber mutations are introduced using standard quickchange mutation protocols (Stratagene; La Jolla, California). Constructsare sequence verified.

Testing of long-acting relaxin compounds may be done using the STZdiabetic rat model (PCO 08400-209).

Suppression with Para-Acetyl-Phenylalanine (pAcF)

Plasmids (e.g. pt_RLX_BA1_AV13am_p1395 (AXID2381)) were used totransform into the Escherichia coli strain W3110B57 to produceRLX-BA1-AV13pAF W3110 B2 strain of E. coli in which expression of the T7polymerase was under control of an arabinose-inducible promoter.Overnight bacterial cultures were diluted 1:100 into shake flaskscontaining 2×YT culture media and grown at 37° C. to an OD₆₀₀ of ˜0.8.Protein expression was induced by the addition of arabinose (0.2%final), and para-acetyl-phenylalanine (pAcF) to a final concentration of4 mM. Cultures were incubated at 37° C. for 5 hours. Cells were pelletedand resuspended in B-PER lysis buffer (Pierce) 100 ul/OD/ml+10 ug/mlDNase and incubated at 37° C. for 30 min. Cellular material was removedby centrifugation and the supernatant removed. The pellet wasre-suspended in an equal amount of SDS-PAGE protein loading buffer. Allsamples were loaded on a 4-12% PAGE gel with MES and DTT. Methods forpurification of relaxin are known to those of ordinary skill in the artand are confirmed by SDS-PAGE, Western Blot analyses, orelectrospray-ionization ion trap mass spectrometry and the like.

His-tagged mutant relaxin proteins can be purified using methods knownto those of ordinary skill in the art. The ProBond Nickel-ChelatingResin (Invitrogen, Carlsbad, CA) may be used via the standard His-taggedprotein purification procedures provided by the manufacturer. Functionalmeasurements of the proteins may be done through methods known in theart, methods provided within this application and incorporatedreferences, and alternatively an ELISA on live cells can be developed toassess relaxin polypeptides of the invention.

TABLE 3 Analyses of Relaxin Variants RP- SE- SDS- LAL Conc. HPLC HPLCBatch/Description PAGE (EU/mg) at A280 purity purity WT Relaxin³ NRMajor NT 2.4 91.2% NT band migrates near the 6 kDa MW standard 20KPEGylated NR Major 7.7 3.4  100% NT Q1pAF^(1,3,4) band 20K PEGylatedmigrates 3.6 2.4  100% NT A5pAF^(1,3,4) between 20K PEGylated the 49 and16.1  2.4  100% NT R18pAF^(1,3,4) 38 kDa 20K PEGylated MW NT 0.6 99.8%NT E5pAF^(2,3) standards 20K PEGylated 10.5  2.3 99.4% NT V7pAF^(2,3,4)20K PEGylated NT 2.0 99.0% NT A18pAF^(2,3) 20K PEGylated 9.1 2.2 99.4%NT W28pAF^(2,3,4) 20K PEGylated 5.1 3.7 99.5% NT V13pAF^(1,3,4) 20KPEGylated NT 0.6 99.6% NT E5pAF² 20K PEGylated 0.0 1.6 99.5% NT L2pAF¹WT Relaxin³ NR Major 0.1 7.5 85.6% NT band migrates near the 6 kDa MWstandard 20K PEGylated NR Major <0.4  2.3 99.3% 99.3% Q1pAF^(1,3) bandmigrates between the 49 and 38 kDa MW standards  5K PEGylated NR Major5.4 1.5 99.4% 99.8% V13pAF^(1,3) band migrates near the 14 kDa MWstandard 10K PEGylated NR Major 9.8 1.8 99.4% 99.5% V13pAF^(1,3) bandmigrates between the 28 and 17 kDa MW standards 20K PEGylated NR Major14.7  1.9 99.0% 99.5% V13pAF^(1,3) band migrates between the 49 and 38kDa MW standards 30K PEGylated NR Major 8.0 2.1 99.5% 99.8% V13pAF^(1,3)band migrates between the 62 and 49 kDa MW standards Samples not reduced(NR); NT = Not Tested ¹pAcF substitution located on A-chain ²pAcFsubstitution located on B-chain ³Asp to Ala substitution on B-chain(BA1) ⁴used for initial PK

TABLE 4 Relaxin Variant Loss of Activity In vitro Activity In vivoFermentor Fold PEG PK Term In vivo Shake Titer (cell Analytical VariantLoss size HL (hr) pharm flask paste) characterization RLX-A- 17  5K, 2.6 (5K), yes  1 gm/L SDS-PAGE, AV13 10K,   8.7 (10K), conc., LAL, 20K, 13.8 (20K), RP-HPLC, 30K  26.8 (30K) SE-HPLC RLX-A- 12 20K  10.7 20Kyes 720 mg/L SDS-PAGE, AQ1 shows conc., RP-HPLC, efficacy LAL, SE-HPLCRLX-A- 12 20K  12.2 yes SDS-PAGE, AA5 conc., RP-HPLC, LAL RLX-A- 15 20K 13.1 yes SDS-PAGE, BV7 conc., RP-HPLC, LAL RLX-A- 17 20K  yes SDS-PAGE,AL2 conc., RP-HPLC RLX-A 17 20K  yes SDS-PAGE, BE5 conc., RP-HPLC RLX-A-21 20K  12.5 yes SDS-PAGE, AR18 conc., RP-HPLC, LAL RLX- 22 20K  yesSDS-PAGE, BE5 conc., RP-HPLC RLX-A- 48 20K  yes SDS-PAGE, BA18 conc.,RP-HPLC RLX-A- 48 20K  13.9 yes SDS-PAGE, BW28 conc., RP-HPLC, LAL

Example 3

This example details expression of Pro-relaxin polypeptides by E. coli.

E. coli expressed Pro-relaxin as a single chain protein composed of 88amino acids. Upon digestion with trypsin and carboxypeptidase, aconnecting peptide and leader sequence are removed. The resultingpeptide is a small 6 kDa two-chain peptide member of the insulinsuperfamily which consists of a 24 residue A-chain and a 29 residueB-chain. The structural fold is characterized by two peptide chainswhich are held together by disulfide bonds as illustrated in FIG. 4 ,specifically two interchain (Cys11-Cys35, i.e., linking Cys11 in the Achain with Cys11 in the B chain, and Cys24-Cys47, i.e., linking Cys24 inthe A chain with Cys47 in the B chain) and one intrachain (Cys10-Cys15,i.e., linking Cys10 and Cys15 in the A chain) disulfide bonds. Thetertiary structure based on a crystal structure of human relaxin-2revealed a compact fold comprising three helical segments and a shortextended region that enclose a hydrophobic core.

Relaxin with one or more non-naturally encoded amino acid(s) provides aunique chemistry and enables a specific PEGylated recombinant variantcontaining a biosynthetically incorporated, chemically reactive,carbonyl group, by replacement of a natural amino acid withpara-acetylphenylalanine (pAcF), providing a unique covalent site ofattachment for a poly(ethylene) glycol (PEG).

Example 4

This example details expression of Pro-relaxin polypeptides by E coli.

This example describes the scale up of relaxin polypeptide productionusing a five (5) liter fermentor. These methods and scale up may also beused for 10L, 30L, 150L and 1000L batches. In some embodiments of thepresent invention, at least 2 g of relaxin protein is produced for eachliter of cell culture. In another embodiment of the present invention,at least 4 g of relaxin protein is produced for each liter of cellculture. In another embodiment of the present invention, at least 6 g ofrelaxin protein is produced for each liter of cell culture. In anotherembodiment of the present invention, at least 8 g of relaxin protein isproduced for each liter of cell culture. In another embodiment of thepresent invention, at least 10 g of relaxin protein is produced for eachliter of cell culture. In another embodiment of the present invention,at least 15 g of relaxin protein is produced for each liter of cellculture. In another embodiment of the present invention, at least 20 gof relaxin protein is produced for each liter of cell culture.

2.1 Seed Shake Flasks

Escherichia coli strain W3110B57 [F-IN(rrnD-rrnE) lambda-araB::g1 tctAfhuA::dhfr ompT::cat] harboring plasmid pt_RLX_BA1_AV13am_p1395(AXID2381) was used to produce RLX-BA1-AV13pAF. A single research cellbank (RCB) vial was removed from −80° C. and thawed at room temperature,then 50 μL was used to inoculate 50 mL of Seed Media (a chemicallydefined medium) supplemented with 50 μg/mL kanamycin sulfate in a 250 mLbaffled Erlenmeyer flask. The primary seed culture was grown forapproximately 18 hours at 37° C., and 250 rpm (1-inch throw). Theprimary seed culture was sub-cultured into a secondary seed culture toan optical density measured at 600 nm wavelength (OD600) of 0.05 in a500 mL baffled Erlenmeyer flask containing 100 mL of Seed Mediumsupplemented with 50 μg/mL kanamycin sulfate. The secondary seed culturewas grown at 37° C., and 250 rpm (1-inch throw) for approximately 8hours or when the OD600 reached between 2 and 4.

2.2 Fermentors

Sartorius Biostat B 5-L vessels were filled with 2.1-L of ProductionMedia (a chemically defined medium) supplemented with 50 μg/L ofkanamycin sulfate. Secondary seed cultures were used to inoculate thefermentors to an initial OD600 of 0.035. The cultures were grown 37° C.and the dissolved oxygen was set to maintain 30% (air saturation) with aprimary agitation (480-1200 rpm) cascade and a secondary O2 cascade. Anair flow rate of 5 LPM with 6 psi back pressure was maintainedthroughout the fermentation. The pH of the culture was set at 7.2±0.05with the addition of 15% ammonium hydroxide and Dow Chemical P2000antifoam was added as needed for foam control. When the culture reachedan OD600 of between 35±5 (when the initial glycerol in the batch mediumwas nearly depleted), a bolus feed of 200 mL was given initiated and atthe same time the pH set point was adjusted from 7.2 to 6.6. After theinitial bolus feed, a continuous feed was initiated at a rate of 0.25mL/L/min and continued until harvest. Immediately after starting thefeed, 2.5 mL/L (0.2 g/L final culture volume) of a 100 g/L L-Ala-pAcFdipeptide solution made in water was added to the fermentor. Fifteenminutes after dipeptide addition, the culture was induced by addingL-arabinose (recipe given in PTR-FGF-002) to a concentration of 2 g/L(final culture volume). The culture was grown 6 hours after arabinoseaddition and harvested.

FIG. 5 shows an SDS-PAGE gel of the prorelaxin produced by these methodswith a chain B1 amino acid as Ala and a para-acetyl phenylalanine in the13^(th) amino acid position of the A chain, substituted for valine.

Example 5

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a relaxinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 5,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22 (i.e., at the carboxyl terminus of theprotein of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs:3, 5, 7, 9, 11) and each of the residues before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 (i.e., at thecarboxyl terminus of the protein of SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NOs: 4, 6, 8, 10, 12), or any other position in arelaxin, relaxin variant, or prorelaxin containing said relaxin orrelaxin variant, is separately substituted with a non-naturally encodedamino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into relaxin are SEQ ID NO: 1 and 2, or SEQ IDNO: 4 and 5 or 6 (A and B chains of relaxin), and SEQ ID NO: 17 or 18(muttRNA, M. jannaschii), and 16, 30, 31 or 32 (TyrRS LW1, 5, or 6)described in Example 2 above.

Once modified, the relaxin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:R-PEG(N)—O—(CH2)n-O—NH2where R is methyl, n is 3 and N is approximately 5,000 MW. The purifiedrelaxin containing p-acetylphenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, MO) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-relaxin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 6

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a relaxinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 20,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54 (i.e., at the carboxyl terminus of the protein of SEQ ID NO:1 or the corresponding amino acids in SEQ ID NOs: 2 and 3), or any otherposition in a relaxin, relaxin variant, or prorelaxin containing saidrelaxin or relaxin variant, is separately substituted with anon-naturally encoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into relaxin are SEQ ID NO: 4 and 5 or 6 (A and Bchains of relaxin), and SEQ ID NO: 17 or 18 (muttRNA, M. jannaschii),and sequences described above and incorporated for site-specificincorporation of p-acetyl-phenylalanine.

Once modified, the relaxin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:R-PEG(N)—O—(CH2)n-O—NH2where R is methyl, n is 3 and N is approximately 20,000 MW. The purifiedrelaxin containing p-acetyl-phenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, MO) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-relaxin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 7

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a relaxinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 20,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53 (i.e., at the carboxyl terminus of the protein of SEQ ID NO: 1 orthe corresponding amino acid positions in SEQ ID NOs: 2 and 3), or anyother position in a relaxin, relaxin variant, or prorelaxin containingsaid relaxin or relaxin variant, is separately substituted with anon-naturally encoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into relaxin are SEQ ID NO: 4 and 5 or 6 (A and Bchains of relaxin), and SEQ ID NO: 17 or 18 (muttRNA, M. jannaschii),and sequences described above and incorporated for site-specificincorporation of p-acetyl-phenylalanine.

Once modified, the relaxin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:R-PEG(N)—O—(CH2)n-O—NH2where R is methyl, n is 3 and N is approximately 20,000 MW. The purifiedrelaxin containing p-acetyl-phenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, MO) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-relaxin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 8

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a relaxinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 20,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54 (i.e., at the carboxyl terminus of the protein of SEQ ID NO:1 or the corresponding amino acid positions in SEQ ID NOs: 2 and 3), orany other position in a relaxin, relaxin variant, or prorelaxincontaining said relaxin or relaxin variant, is separately substitutedwith a non-naturally encoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into relaxin are SEQ ID NO: 1, or SEQ ID NO: 4and 5 or 6 (A and B chains of relaxin), and SEQ ID NO: 17 or 18(muttRNA, M. jannaschii), and sequences described above and incorporatedfor site-specific incorporation of p-acetyl-phenylalanine.

Once modified, the relaxin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:R-PEG(N)—O—(CH2)n-O—NH2where R is methyl, n is 3 and N is approximately 20,000 MW. The purifiedrelaxin containing p-acetyl-phenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical. St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, MO) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-relaxin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 9

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a relaxinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 30,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54 (i.e., at the carboxyl terminus of the protein of SEQ ID NO:1 or the corresponding positions in SEQ ID NOs: 2 and 3), or any otherposition in a relaxin, relaxin variant, or prorelaxin containing saidrelaxin or relaxin variant, is separately substituted with anon-naturally encoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into relaxin are SEQ ID NO: 1 (or SEQ ID NO: 2,or 3) or SEQ ID NO: 4 and 5 or 6 (A and B chains of relaxin), and SEQ IDNO: 17 or 18 (muttRNA, M. jannaschii), and sequences described above andincorporated for site-specific incorporation of p-acetyl-phenylalanine.

Once modified, the relaxin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:R-PEG(N)—O—(CH2)n-O—NH2where R is methyl, n is 3 and N is approximately 30,000 MW. The purifiedrelaxin containing p-acetyl-phenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, MO) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-relaxin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 10

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a relaxinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 40,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54 (i.e., at the carboxyl terminus of the protein of SEQ ID NO:1 or the corresponding positions in SEQ ID NOs: 2 and 3), or any otherposition in a relaxin, relaxin variant, or prorelaxin containing saidrelaxin or relaxin variant, is separately substituted with anon-naturally encoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into relaxin are SEQ ID NO: 1 or 2), or SEQ IDNO: 4 and 5 or 6 (A and B chains of relaxin) and SEQ ID NO: 17 or 18(muttRNA, M. jannaschii), and sequences described above and incorporatedfor site-specific incorporation of p-acetyl-phenylalanine.

Once modified, the relaxin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:R-PEG(N)—O—(CH2)n-O—NH2where R is methyl, n is 3 and N is approximately 40,000 MW. The purifiedrelaxin containing p-acetyl-phenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, MO) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-relaxin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 11

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a relaxinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 10,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54 (i.e., at the carboxyl terminus of the protein of SEQ ID NO:1 or the corresponding positions in SEQ ID NOs: 2 and 3), or any otherposition in a relaxin, relaxin variant, or prorelaxin containing saidrelaxin or relaxin variant, is separately substituted with anon-naturally encoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into relaxin are SEQ ID NO: 1, or 2 or SEQ ID NO:4 and 5 or 6 (A and B chains of relaxin), and SEQ ID NO: 17 or 18(muttRNA, M. jannaschii), and sequences described above and incorporatedfor site-specific incorporation of p-acetyl-phenylalanine.

Once modified, the relaxin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:R-PEG(N)—O—(CH2)n-O—NH2

where R is methyl, n is 3 and N is approximately 10,000 MW. The purifiedrelaxin containing p-acetyl-phenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, MO) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks. W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-relaxin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 12

Conjugation with a PEG consisting of a hydroxylamine group linked to thePEG via an amide linkage.

A PEG reagent having the following structure is coupled to aketone-containing non-naturally encoded amino acid using the proceduredescribed in Examples 3-9:R-PEG(N)—O—(CH₂)2-NH—C(O)(CH₂)—O—NH₂where R=methyl, n=4 and N is approximately 5,000 MW-40,000 MW. Thereaction, purification, and analysis conditions are as described andknown in the art.

Example 13

This example details the introduction of two distinct non-naturallyencoded amino acids into relaxin polypeptides and relaxin analogpolypeptides.

This example demonstrates a method for the generation of a relaxinpolypeptide that incorporates non-naturally encoded amino acidcomprising a ketone functionality at two positions among the followingresidues: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 (i.e., at the carboxylterminus of the protein of SEQ ID NO: 1 or the corresponding positionsin SEQ ID NOs: 2 and 3), or any other position in a relaxin, relaxinvariant, or prorelaxin containing said relaxin or relaxin variant. Therelaxin polypeptide is prepared as described above, except that theselector codon is introduced at two distinct sites within the nucleicacid.

Example 14

This example details conjugation of relaxin polypeptide or relaxinanalog polypeptide to a hydrazide-containing PEG and subsequent in situreduction.

A relaxin polypeptide incorporating a carbonyl-containing amino acid isprepared according to the procedure described above. Once modified, ahydrazide-containing PEG having the following structure is conjugated tothe relaxin polypeptide:R-PEG(N)—O—(CH2)2-NH—C(O)(CH2)n-X—NH—NH2where R=methyl, n=2 and N=5,000; 10,000, 20,000; 30,000; or 40,000 MWand X is a carbonyl (C═O) group. The purified relaxin containingp-acetylphenylalanine is dissolved at between 0.1-10 mg/mL in 25 mM MES(Sigma Chemical, St. Louis, MO) pH 6.0, 25 mM Hepes (Sigma Chemical, St.Louis, MO) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, MO) pH 4.5, is reacted with a 1 to 100-fold excess ofhydrazide-containing PEG, and the corresponding hydrazone is reduced insitu by addition of stock 1M NaCNBH3 (Sigma Chemical, St. Louis, MO),dissolved in H₂O, to a final concentration of 10-50 mM. Reactions arecarried out in the dark at 4° C. to RT for 18-24 hours. Reactions arestopped by addition of 1 M Tris (Sigma Chemical. St. Louis, MO) at aboutpH 7.6 to a final Tris concentration of 50 mM or diluted intoappropriate buffer for immediate purification.

Example 15

This example details conjugation of relaxin polypeptide or relaxinanalog polypeptide to a hydrazide-containing PEG and subsequent in situreduction.

A relaxin polypeptide incorporating a carbonyl-containing amino acid isprepared according to the procedure described above. Once modified, ahydrazide-containing PEG having the following structure is conjugated tothe relaxin polypeptide:R-PEG(N)—O—(CH2)2-NH—C(O)(CH2)n-X—NH—NH2where R=methyl, n=2 and N=20,000 MW and X is a carbonyl (C═O) group. Thepurified relaxin containing p-acetylphenylalanine is dissolved atbetween 0.1-10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, MO) pH6.0, 25 mM Hepes (Sigma Chemical, St. Louis, MO) pH 7.0, or in 10 mMSodium Acetate (Sigma Chemical, St. Louis, MO) pH 4.5, is reacted with a1 to 100-fold excess of hydrazide-containing PEG, and the correspondinghydrazone is reduced in situ by addition of stock 1M NaCNBH3 (SigmaChemical, St. Louis, MO), dissolved in H2O, to a final concentration of10-50 mM. Reactions are carried out in the dark at 4° C. to RT for 18-24hours. Reactions are stopped by addition of 1 M Tris (Sigma Chemical,St. Louis, MO) at about pH 7.6 to a final Tris concentration of 50 mM ordiluted into appropriate buffer for immediate purification.

Example 16

This example details introduction of an alkyne-containing amino acidinto a relaxin polypeptide or relaxin analog polypeptide andderivatization with mPEG-azide.

The following residues, before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 (i.e., at thecarboxyl terminus of the protein of SEQ ID NO: 1 or the correspondingpositions in SEQ ID NOs: 2 and 3), or any other position in a relaxin,relaxin variant, or prorelaxin containing said relaxin or relaxinvariant, are each substituted with the following non-naturally encodedamino acid:

The sequences utilized for site-specific incorporation ofp-propargyl-tyrosine into relaxin are SEQ ID NO: 1 (or correspondingpositions in SEQ ID NO:2 or 3), SEQ ID NO: 17 or 18 (muttRNA, M.jannaschii), and 23, 24 or 25 described above. The relaxin polypeptidecontaining the propargyl tyrosine is expressed in E. coli and purifiedusing the conditions described above.

The purified relaxin containing propargyl-tyrosine dissolved at between0.1-10 mg/mL in PB buffer (100 mM sodium phosphate, 0.15 M NaCl, pH=8)and a 10 to 1000-fold excess of an azide-containing PEG is added to thereaction mixture. A catalytic amount of CuSO4 and Cu wire are then addedto the reaction mixture. After the mixture is incubated (including butnot limited to, about 4 hours at room temperature or 37° C., orovernight at 4° C.), H2O is added and the mixture is filtered through adialysis membrane. The sample can be analyzed for the addition,including but not limited to, by similar procedures described in Example3. In this Example, the PEG will have the following structure:R-PEG(N)—O—(CH2)2-NH—C(O)(CH2)n-N3where R is methyl, n is 4 and N=5,000; 10,000, 20,000; 30,000; or 40,000MW.

Example 17

This example details substitution of a large, hydrophobic amino acid ina relaxin polypeptide with propargyl tyrosine.

A Phe, Trp or Tyr residue present within one the following regions ofrelaxin: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 (i.e., at the carboxylterminus of the protein of SEQ ID NO: 1 or the corresponding positionsin SEQ ID NOs: 2 and 3), or any other position in a relaxin, relaxinvariant, or prorelaxin containing said relaxin or relaxin variant, issubstituted with the following non-naturally encoded amino acid asdescribed above:

Once modified, a PEG is attached to the relaxin polypeptide variantcomprising the alkyne-containing amino acid. The PEG will have thefollowing structure:Me-PEG(N)—O—(CH2)2-N3and coupling procedures would follow those in examples above. This willgenerate a relaxin polypeptide variant comprising a non-naturallyencoded amino acid that is approximately isosteric with one of thenaturally-occurring, large hydrophobic amino acids and which is modifiedwith a PEG derivative at a distinct site within the polypeptide.

Example 18

This example details generation of a relaxin polypeptide homodimer,heterodimer, homomultimer, or heteromultimer separated by one or morePEG linkers. Relaxin polypeptide multimers may be formed betweenprorelaxins or between mature A and B chain relaxin polypeptides of theinvention.

The alkyne-containing relaxin polypeptide variant produced in theexample above is reacted with a bifunctional PEG derivative of the form:N3-(CH2)n-C(O)—NH—(CH2)2-O-PEG(N)—O—(CH2)2-NH—C(O)4CH2)n-N3where n is 4 and the PEG has an average MW of approximately 5,000;10,000; 20,000; 30,000; or 40,000 MW to generate the correspondingrelaxin polypeptide homodimer where the two relaxin molecules arephysically separated by PEG. In an analogous manner a relaxinpolypeptide may be coupled to one or more other polypeptides to formheterodimers, homomultimers, or heteromultimers. Coupling, purification,and analyses will be performed as in the examples above.

Example 19

This example details generation of a relaxin polypeptide homodimer,heterodimer, homomultimer, or heteromultimer separated by one or morePEG linkers. Relaxin polypeptide multimers may be formed between Achains and other A chains or B chains and other B chains.

The alkyne-containing relaxin polypeptide variant produced in theexample above is reacted with a bifunctional PEG derivative of the form:N3-(CH2)n-C(O)—NH4CH2)2-O-PEG(N)—O—(CH2)2-NH—C(O)—(CH2)n-N3where n is 4 and the PEG has an average MW of approximately 5,000;10,000; 20,000; 30,000; or 40,000 MW to generate the correspondingrelaxin polypeptide homodimer where the two relaxin molecules arephysically separated by PEG. In an analogous manner a relaxinpolypeptide may be coupled to one or more other polypeptides to formheterodimers, homomultimers, or heteromultimers. Coupling, purification,and analyses will be performed as in the examples above.

Example 20

This example details coupling of a saccharide moiety to a relaxinpolypeptide.

One residue of the following is substituted with the non-naturallyencoded amino acid below: before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 (i.e., at thecarboxyl terminus of the protein of SEQ ID NO: 1 or the correspondingpositions in SEQ ID NOs: 2 and 3), or any other position in a relaxin,relaxin variant, or prorelaxin containing said relaxin or relaxinvariant, as described above.

Once modified, the relaxin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with a β-linked aminooxyanalogue of N-acetylglucosamine (GlcNAc). The relaxin polypeptidevariant (10 mg/mL) and the aminooxy saccharide (21 mM) are mixed inaqueous 100 mM sodium acetate buffer (pH 5.5) and incubated at 37° C.for 7 to 26 hours. A second saccharide is coupled to the firstenzymatically by incubating the saccharide-conjugated relaxinpolypeptide (5 mg/mL) with UDP-galactose (16 mM) andβ-1,4-galacytosyltransferase (0.4 units/mL) in 150 mM HEPES buffer (pH7.4) for 48 hours at ambient temperature (Schanbacher et al. J. Biol.Chem. 1970, 245, 5057-5061).

Example 21A

This example details generation of a PEGylated relaxin polypeptideantagonist.

A residue, including but not limited to, those involved in relaxinreceptor binding is substituted with the following non-naturally encodedamino acid as described above. Once modified, the relaxin polypeptidevariant comprising the carbonyl-containing amino acid will be reactedwith an aminooxy-containing PEG derivative of the form:R-PEG(N)—O—(CH2)n-O—NH2where R is methyl, n is 4 and N is 5,000; 10,000; 20,000; 30,000; or40,000 MW to generate a relaxin polypeptide antagonist comprising anon-naturally encoded amino acid that is modified with a PEG derivativeat a single site within the polypeptide. Coupling, purification, andanalyses are performed as described above.

Example 21B

Generation of a relaxin polypeptide homodimer, heterodimer,homomultimer, or heteromultimer in which the relaxin molecules arelinked directly

A relaxin polypeptide variant comprising the alkyne-containing aminoacid can be directly coupled to another relaxin polypeptide variantcomprising the azido-containing amino acid. In an analogous manner arelaxin polypeptide may be coupled to one or more other polypeptides toform heterodimers, homomultimers, or heteromultimers. More descriptionregarding multimers which may be formed is provided above in Examples 16and 17 and coupling, purification, and analyses are performed asdescribed above.

Example 22

The polyalkylene glycol (P—OH) is reacted with the alkyl halide (A) toform the ether (B). In these compounds, n is an integer from one to nineand R′ can be a straight- or branched-chain, saturated or unsaturatedC1, to C20 alkyl or heteroalkyl group. R′ can also be a C3 to C7saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, asubstituted or unsubstituted aryl or heteroaryl group, or a substitutedor unsubstituted alkaryl (the alkyl is a C1 to C20 saturated orunsaturated alkyl) or heteroalkaryl group. Typically, PEG-OH ispolyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG)having a molecular weight of 800 to 40,000 Daltons (Da).

Example 23

mPEG-OH+Br—CH₂—C≡CH→mPEG-O—CH₂—C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Asolution of propargyl bromide, dissolved as an 80% weight solution inxylene (0.56 mL, 5 mmol, 50 equiv., Aldrich), and a catalytic amount ofKI were then added to the solution and the resulting mixture was heatedto reflux for 2 hours. Water (1 mL) was then added and the solvent wasremoved under vacuum. To the residue was added CH2Cl2 (25 mL) and theorganic layer was separated, dried over anhydrous Na2SO4, and the volumewas reduced to approximately 2 mL. This CH2Cl2 solution was added todiethyl ether (150 mL) drop-wise. The resulting precipitate wascollected, washed with several portions of cold diethyl ether, and driedto afford propargyl-O-PEG.

Example 24

mPEG-OH+Br—(CH₂)₃—C≡CH→mPEG-O—(CH₂)₃—C≡CH

The mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g,0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL).Fifty equivalents of 5-bromo-1-pentyne (0.53 mL, 5 mmol, Aldrich) and acatalytic amount of KI were then added to the mixture. The resultingmixture was heated to reflux for 16 hours. Water (1 mL) was then addedand the solvent was removed under vacuum. To the residue was addedCH2Cl2 (25 mL) and the organic layer was separated, dried over anhydrousNa2SO4, and the volume was reduced to approximately 2 mL. This CH2Cl2solution was added to diethyl ether (150 mL) drop-wise. The resultingprecipitate was collected, washed with several portions of cold diethylether, and dried to afford the corresponding alkyne. 5-chloro-1-pentynemay be used in a similar reaction.

Example 25

m-HOCH₂C₆H₄OH+NaOH+Br—CH₂—C≡CH→m-HOCH₂C₆H₄O—CH₂—C≡CH  (1)m-HOCH₂C₆H₄O—CH₂—C≡CH+MsCl+N(Et)₃ →m-MsOCH₂C₆H₄O—CH₂—C≡CH  (2)m-MsOCH₂C₆H₄O—CH₂—C≡CH+LiBr→m-Br—CH₂C₆H₄O—CH₂—C≡CH  (3)mPEG-OH+m-Br—CH₂C₆H₄O—CH₂—C≡CH→mPEG-O—CH₂—C₆H₄O—CH₂—C≡CH  (4)

To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL)and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g,37.5 mmol) and then a solution of propargyl bromide, dissolved as an 80%weight solution in xylene (3.36 mL, 30 mmol). The reaction mixture washeated at reflux for 6 hours. To the mixture was added 10% citric acid(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over MgSO4 andconcentrated to give the 3-propargyloxybenzyl alcohol.

Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of compound 3 (2.0 g, 11.0 mmol) inCH2Cl2 at 0° C. and the reaction was placed in the refrigerator for 16hours. A usual work-up afforded the mesylate as a pale yellow oil. Thisoil (2.4 g, 9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0mmol) was added. The reaction mixture was heated to reflux for 1 hourand was then cooled to room temperature. To the mixture was added water(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa2SO4, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in THF (20 mL)and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) wasadded with vigorous stirring over a period of several minutes followedby addition of the bromide obtained from above (2.55 g, 11.4 mmol) and acatalytic amount of KI. The cooling bath was removed and the resultingmixture was heated to reflux for 12 hours. Water (1.0 mL) was added tothe mixture and the solvent was removed under vacuum. To the residue wasadded CH2Cl2 (25 mL) and the organic layer was separated, dried overanhydrous Na2SO4, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in a whiteprecipitate, which was collected to yield the PEG derivative.

Example 26

mPEG-NH₂+X—C(O)—(CH₂)_(n)—C≡CR′→mPEG-NH—C(O)—(CH₂)_(n)—C≡CR′

The terminal alkyne-containing poly(ethylene glycol) polymers can alsobe obtained by coupling a poly(ethylene glycol) polymer containing aterminal functional group to a reactive molecule containing the alkynefunctionality as shown above, n is between 1 and 10. R′ can be H or asmall alkyl group from C1 to C4.

Example 27

HO₂C—(CH₂)₂—C≡CH+NHS+DCC→NHSO—C(O)—(CH₂)₂—C≡CH  (1)mPEG-NH₂+NHSO—C(O)—(CH₂)₂—C≡CH→mPEG-NH—C(O)—(CH₂)₂—C≡CH  (2)

4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH2Cl2 (25 mL).N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) wereadded and the solution was stirred overnight at room temperature. Theresulting crude NHS ester 7 was used in the following reaction withoutfurther purification.

mPEG-NH2 with a molecular weight of 5,000 Da (mPEG-NH2, 1 g, Sunbio) wasdissolved in THF (50 mL) and the mixture was cooled to 4° C. NHS ester 7(400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. Themixture was allowed to stir for 3 hours while warming to roomtemperature. Water (2 mL) was then added and the solvent was removedunder vacuum. To the residue was added CH2Cl2 (50 mL) and the organiclayer was separated, dried over anhydrous Na2SO4, and the volume wasreduced to approximately 2 mL. This CH2Cl2 solution was added to ether(150 mL) drop-wise. The resulting precipitate was collected and dried invacuo.

Example 28

This Example represents the preparation of the methane sulfonyl ester ofpoly(ethylene glycol), which can also be referred to as themethanesulfonate or mesylate of poly(ethylene glycol). The correspondingtosylate and the halides can be prepared by similar procedures.mPEG-OH+CH₃SO₂Cl+N(Et)₃ →mPEG-O—SO₂CH₃ →mPEG-N₃

The mPEG-OH (MW=3,400, 25 g, 10 mmol) in 150 mL of toluene wasazeotropically distilled for 2 hours under nitrogen and the solution wascooled to room temperature. 40 mL of dry CH2Cl2 and 2.1 mL of drytriethylamine (15 mmol) were added to the solution. The solution wascooled in an ice bath and 1.2 mL of distilled methanesulfonyl chloride(15 mmol) was added dropwise. The solution was stirred at roomtemperature under nitrogen overnight, and the reaction was quenched byadding 2 mL of absolute ethanol. The mixture was evaporated under vacuumto remove solvents, primarily those other than toluene, filtered,concentrated again under vacuum, and then precipitated into 100 mL ofdiethyl ether. The filtrate was washed with several portions of colddiethyl ether and dried in vacuo to afford the mesylate.

The mesylate (20 g, 8 mmol) was dissolved in 75 ml of THF and thesolution was cooled to 4° C. To the cooled solution was added sodiumazide (1.56 g, 24 mmol). The reaction was heated to reflux undernitrogen for 2 hours. The solvents were then evaporated and the residuediluted with CH2Cl2 (50 mL). The organic fraction was washed with NaClsolution and dried over anhydrous MgSO4. The volume was reduced to 20 mland the product was precipitated by addition to 150 ml of cold dryether.

Example 29

N₃—C₆H₄—CO₂H→N₃—C₆H₄CH₂OH  (1)N₃—C₆H₄CH₂OH→Br—CH₂—C₆H₄—N₃  (2)mPEG-OH+Br—CH₂—C₆H₄—N₃ →mPEG-O—CH₂—C₆H₄—N₃  (3)

4-azidobenzyl alcohol can be produced using the method described in U.S.Pat. No. 5,998,595, which is incorporated by reference herein.Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of 4-azidobenzyl alcohol (1.75 g, 11.0mmol) in CH2Cl2 at 0° C. and the reaction was placed in the refrigeratorfor 16 hours. A usual work-up afforded the mesylate as a pale yellowoil. This oil (9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g,23.0 mmol) was added. The reaction mixture was heated to reflux for 1hour and was then cooled to room temperature. To the mixture was addedwater (2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa2SO4, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg,0.5 mmol) in THF (35 mL) and the bromide (3.32 g, 15 mmol) was added tothe mixture along with a catalytic amount of KI. The resulting mixturewas heated to reflux for 12 hours. Water (1.0 mL) was added to themixture and the solvent was removed under vacuum. To the residue wasadded CH2Cl2 (25 mL) and the organic layer was separated, dried overanhydrous Na2SO4, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in aprecipitate, which was collected to yield mPEG-O—CH2-C6H4-N3.

Example 30

NH₂—PEG-O—CH₂CH₂CO₂H+N₃—CH₂CH₂CO₂—NHS+N₃—CH₂CH₂—C(O)NH-PEG-O—CH₂CH₂CO₂H

NH2-PEG-O—CH2CH2CO2H (MW 3,400 Da, 2.0 g) was dissolved in a saturatedaqueous solution of NaHCO3 (10 mL) and the solution was cooled to 0° C.3-azido-1-N-hydroxysuccinimido propionate (5 equiv.) was added withvigorous stirring. After 3 hours, 20 mL of H2O was added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H2SO4 and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH2Cl2(100 mL×3), dried over Na2SO4 and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the omega-carboxy-azide PEG derivative.

Example 31

mPEG-OMs+HC≡CLi→mPEG-O—CH₂—CH₂—C≡C—H

To a solution of lithium acetylide (4 equiv.), prepared as known in theart and cooled to −78° C. in THF, is added dropwise a solution ofmPEG-OMs dissolved in THE with vigorous stirring. After 3 hours, thereaction is permitted to warm to room temperature and quenched with theaddition of 1 mL of butanol. 20 mL of H2O is then added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H2SO4 and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH2Cl2(100 mL×3), dried over Na2SO4 and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the 1-(but-3-ynyloxy)-methoxypolyethylene glycol(mPEG).

Example 32

Azide- and acetylene-containing amino acids can be incorporatedsite-selectively into proteins using the methods described in L. Wang,et al., (2001), Science 292:498-500, J. W. Chin et al., Science301:964-7 (2003)), J. W. Chin et al., (2002), Journal of the AmericanChemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002),Chem Bio Chem 3(11):1135-1137; J. W. Chin, et al., (2002), PNAS UnitedStates of America 99:11020-11024: and, L. Wang, & P. G. Schultz, (2002).Chem. Comm., 1:1-11. Once the amino acids were incorporated, thecycloaddition reaction is carried out with 0.01 mM protein in phosphatebuffer (PB), pH 8, in the presence of 2 mM PEG derivative, 1 mM CuSO4,and ˜1 mg Cu-wire for 4 hours at 37° C.

Example 33

This example describes the synthesis of p-Acetyl-D,L-phenylalanine (pAF)and m-PEG-hydroxylamine derivatives.

The racemic pAF is synthesized using the previously described procedurein Zhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. & Schultz,P. G., Biochemistry, (2003) 42, 6735-6746.

To synthesize the m-PEG-hydroxylamine derivative, the followingprocedures are completed. To a solution of (N-t-Boc-aminooxy)acetic acid(0.382 g 2.0 mmol) and 1,3-Diisopropylcarbodiimide (0.16 mL, 1.0 mmol)in dichloromethane (DCM, 70 mL), which is stirred at room temperature(RT) for 1 hour, methoxy-polyethylene glycol amine (m-PEG-NH2, 7.5 g,0.25 mmol, Mt. 30 K, from BioVectra) and Diisopropylethylamine (0.1 mL,0.5 mmol) is added. The reaction is stirred at RT for 48 hours, and thenis concentrated to about 100 mL. The mixture is added dropwise to coldether (800 mL). The t-Boc-protected product precipitated out and iscollected by filtering, washed by ether 3×100 mL. It is further purifiedby re-dissolving in DCM (100 mL) and precipitating in ether (800 mL)twice. The product is dried in vacuum yielding 7.2 g (96%), confirmed byNMR and Nihydrin test.

The deBoc of the protected product (7.0 g) obtained above is carried outin 50% TFA/DCM (40 mL) at 0° C. for 1 hour and then at RT for 1.5 hour.After removing most of TFA in vacuum, the TFA salt of the hydroxylaminederivative is converted to the HCl salt by adding 4N HCl in dioxane (1mL) to the residue. The precipitate is dissolved in DCM (50 mL) andre-precipitated in ether (800 mL). The final product (6.8 g, 97%) iscollected by filtering, washed with ether 3×100 mL, dried in vacuum,stored under nitrogen. Other PEG (5K, 20K) hydroxylamine derivatives aresynthesized using the same procedure.

Example 34

In Vivo Studies of PEGylated Relaxin

PEG-Relaxin, unmodified relaxin and buffer solution are administered tomice or rats. The results will show superior activity and prolonged halflife of the PEGylated relaxin of the present invention compared tounmodified relaxin. Similarly, modified relaxin, unmodified relaxin, andbuffer solution are administered to mice or rats.

Pharmacokinetic Analysis

A relaxin polypeptide of the invention is administered by intravenous orsubcutaneous routes to mice. The animals are bled prior to and at timepoints after dosing. Plasma is collected from each sample and analyzedby radioimmunoassay. Elimination half-life can be calculated andcompared between relaxin polypeptides comprising a non-naturally encodedamino acid and wild-type relaxin or various relaxin analog polypeptidesof the invention. Similarly, relaxin polypeptides of the invention maybe administered to cynomolgus monkeys. The animals are bled prior to andat time points after dosing. Plasma is collected from each sample andanalyzed by radioimmunoassay.

The polypeptide may be administered the mice via multiple doses,continuous infusion, or a single dose, etc.

Example 35

Relaxin is expressed using Novagen expression system (inducible T7promoter; described in detail in the pET System Manual, version 9,hereby incorporated by reference), expression vector pET30a andexpression strain BL21(DE3).

2 mL of LB/Kanamycin (10 μg/ml) culture are inoculated with a sweep fromBL21 (DE3) plate transformed with the desired analog. This decreaseseffects caused by colony to colony variability in expression levels.This culture is grown overnight at 37′C with vigorous shaking and thefollowing day, 10 ml LB/Kanamycin culture is inoculated with 1 ml fromthe overnight culture (OD600˜0.4-0.5). The remaining mL of the overnightculture may be frozen as glycerol stock.

10 mL of the grown culture is put at 37° C. and 250 rpm for 30-45 minuntil OD600 reaches 0.8-0.9. This is then induced with 1 mM IPTG (with 1mL that may be set aside as non-induced culture control) and harvestedusually 3-4 hours post-induction and analyzed on SDS-PAGE.

It is also possible to do a time-course of expression (e.g. points1,2,4,6 hours post-induction and O/N) to determine the rate ofaccumulation, protein stability, etc.

Gel Analysis: at a desired time point post-induction 1 mL is harvestedfrom the culture, the cells are spun down, resuspended in 100 μl of2×SDS-PAGE, sonicated to reduce viscosity and 10 μl are run on SDS-PAGE.If desired, this can be compared to non-induced control or controlsand/or known positive control or standard and expression level may beestimated (e.g. good expression could be at >100 g/ml). Western blotanalysis may also be used. It is also possible to set aside 4 ml of thecultures, prepare inclusion bodies (if expressing insoluble analogs) andobtain mass spec analysis on these to confirm the identity of theover-expressed protein.

For larger scale protein expression, >250 mL of LB/Kanamycin (10 μg/ml)are inoculated with 250 μL of frozen glycerol stock and grown overnight.The following day, 10×1 L LB/Kanamycin cultures are inoculated with 25mL from the overnight culture (OD600˜0.1).

1 L cultures are grown at 37° C., and 250 rpm˜2 h until OD600 reaches0.8-0.9. This is then induced with 1 mM IPTG and harvested 4 hpost-induction or the following morning (harvest may use centrifugationfor 15 min at 4,000 rpm). The pellets are rinsed with 50 mM Tris-HCl, pH8.0 (50 ml per pellet+50 ml to rinse the bottle) if it is desired toreduce endotoxin and facilitate purification. Pellets are pooledtogether and spun again.

Example 36

Pichia Expression Study—DNA Prep, Electroporation, Expression Protocols

This example provides a protocol for the preparation of relaxinpolypeptides of the present invention in Pichia. A plasmid can be usedfor cloning into Pichia and this or other modified plasmids may be usedto obtain protein expression of relaxin polypeptides in Pichia,modifications made to the plasmid using methods known in the art.

On day 1 of the protocol, there is an overnight digestion, typicallyusing 2U enzyme per μg DNA to be digested and 10 mL YPhyD culture isinoculated overnight in a 50 mL flask, shaking at 260 rpm at 30° C. fromthe glycerol stock.

DNA Preparation

DNA is precipitated by the addition first of 1/10^(th) volume sterile 3MNaOAc and then of 0.7 volumes sterile IPA and then the sample isvigorously mixed and the precipitation is continued overnight at −20°C., or at −70° C. until frozen. The DNA is then pelleted bycentrifugation (benchtop centrifuge 14,000 rpm/10 minutes), supernatantremoved, and the pellet is washed using 500 μL of sterile 70% ETOH. Spin(bench-top centrifuge 14,000 rpm/10 minutes) and decant supernatant andair dry pellet for 15-20 minutes. Resuspend DNA pellet with sterilewater to 1 μg/μl and transform Pichia with 10 μg DNA.

Electroporation

Using overnight culture with OD₆₀₀, dilute in YPhyD to OD₆₀₀=0.2. Shakeculture at 260 rpm at 30° C. until OD₆₀₀ reaches 0.8-1.0. Collect cellsby centrifugation (4000 rpm/5 minutes). Decant medium, wash cells in 20mL ice cold sterile water, decant again and repeat. After water wash,wash pellet in 20 mL of ice-cold sterile 1 M sorbitol, decant, andresuspend washed cell pellet in 600 μL of 1 M cold sorbitol, then thismay be stored on ice.

From the washed cells, mix 50 μL with 10 μg linearized DNA in sterile1.5 mL eppendorf tube, mix gently and incubate on ice for 25 minutes.Transfer cell/DNA mixture to prechilled 0.2 cm cuvette using longpipette tips. Electroporate cells using BioRad GenePulsar II unit withthe following settings: 2000 V, 200 Ohms, 25 μFd (use single pulse) andimmediately add 0.5 mL YPhyD medium to the cuvette and mix by pipetting.Transfer entire contents to sterile round bottom tube and shake gently(200 rpm) for 30 minutes at 30° C. Plate and spread cells evenly andincubate plates, inverted, for 3 days at 30° C.

After three day incubation, pick colonies with a loop and inoculate 10ml BYPhyD media in a 50 ml flask and incubate for 3 days at 30° C. Countthe colonies on the 20 μl plates and record the average number and thenharvest cells, first by preparing 2 sets of cryovials labeled withstrain name and clone number, relaxin (i.e. protein expressed), anddate. Transfer culture to 15 ml conical tube, take OD₆₀₀ of eachculture, dilute culture 1:50 or 1:20 in YPhyD medium. Save an alquot ofculture for glycerol stock. Then pellet yeast at 4000 rpm for 5 min atRT, transfer the supernatant to a new, labeled 15 mL conical tube, andstore at −20 or −80° C. until needed for analytical data.

Protein Expression Analysis

Run samples on 4-12% NuPAGE TB gel (Novex). SDS-PAGE reagents used fromInvitrogen, analyze by Western blot or Stained-gel analysis

Media Formulations

Buffered Yeast Phytone Dextrose (BYPhyD)

Yeast Extract 10 g/L Phytone Peptone 20 g/L 1M potassium phosphatebuffer (pH 6) 100 ml/L 10X YNB 100 ml/L 20% Dextrose 100 ml/LYeast Phytone Dextrose (YPhyD)

Yeast Extract 10 g/L Phytone Peptone 20 g/L 20% Dextrose 100 ml/L10×YNB (13.4% Yeast Nitrogen Base with Ammonium Sulfate without AminoAcids)

Yeast Nitrogen Base 134 g/L

Example 37

Relaxin A21G Production

In this example, 4.0 L culture were fermented to produce 13.4 g wet cellpaste and an inclusion body preparation was performed with and withoutTriton-X100. 2.07 g wet inclusion bodies were produced in this manner,and solubilization and refolding followed. The inclusion bodies wereresuspended with 200 mL H₂O per gram of wet inclusion bodies (IBs) to afinal concentration of 3 mM and cysteine is added to the resuspension.IB's are then solubilized by pH increase to 11.5 for 1 hour at RT.Refolding was then allowed to occur by dropping the pH of thesolubilized material to 10.6±0.1 and stored at 2-8° C. for ˜72 hours andthese results are shown in FIG. 10A-10B. The refold reaction was stoppedby addition of HCl to a final pH of 3.0, 0.45 μM filtered and stored at2-8° C. until further processing.

The refolded protein was purified by increasing the pH of quenchedrefold to 8.0 with Tris base and directly loading onto a Q HP column.Conductivity of load in the instance shown was >3.5 mS/cm. Runconditions were (A) 20 mM Tris, 8.0; (B) 20 mM Tris, 8.0; 200 mM NaCland there was 0-100% B over 30 CV. The correctly refolded proinsulin waspooled and 79 mg proinsulin was recovered.

Ultrafiltration/diafiltration (UF/DF) was done and precipitation wasperformed with 25 mM zinc, precipitated protein was resuspended toconcentration of 2 mg/mL with 20 mM NaOAc, 4.0, 30% ACN, 5 mM EDTA and20K PEG was added to a final molar ratio of 10:1 PEG to protein andallowed to incubate for 48-72 hours at 28° C.

PEG reaction was diluted 1:10 in 0.5×PEG buffer A. 0.22 μM filtered andrun over an SP 650S column. The run conditions were (A) 10 mM NaOAc,4.0, 1 mM EDTA; (B) 10 mM NaOAc, 4.0, 1 mM EDTA, 0.4M NaCl; 0-50% B over20 CV and PEG samples formulated in 10 mM NaCitrate, 6.5: 150 mM NaCland this is shown in FIG. 12A-12B.

These methods were used to produce a variety of relaxin polypeptideswith non-natural amino acids and a range of 0.1-22 mg for the endprotein amounts of the purified and PEGylated variants. ACN was found tohelp solubilize PEG/protein mixture in PEG reaction and zincprecipitation at p1 facilitated concentrating in the presence of CAN.

Example 38

Human Clinical Trial of the Safety and/or Efficacy of PEGylated RelaxinComprising a Non-Naturally Encoded Amino Acid

Objective: To observe the safety and pharmacokinetics of subcutaneouslyadministered PEGylated recombinant human relaxin comprising anon-naturally encoded amino acid.

Patients Eighteen healthy volunteers ranging between 20-40 years of ageand weighing between 60-90 kg are enrolled in the study. The subjectswill have no clinically significant abnormal laboratory values forhematology or serum chemistry, and a negative urine toxicology screen,HIV screen, and hepatitis B surface antigen. They should not have anyevidence of the following: hypertension; a history of any primaryhematologic disease; history of significant hepatic, renal,cardiovascular, gastrointestinal, genitourinary, metabolic, neurologicdisease; a history of anemia or seizure disorder; a known sensitivity tobacterial or mammalian-derived products, PEG, or human serum albumin;habitual and heavy consumer to beverages containing caffeine;participation in any other clinical trial or had blood transfused ordonated within 30 days of study entry; had exposure to relaxin withinthree months of study entry; had an illness within seven days of studyentry; and have significant abnormalities on the pre-study physicalexamination or the clinical laboratory evaluations within 14 days ofstudy entry. All subjects are evaluable for safety and all bloodcollections for pharmacokinetic analysis are collected as scheduled. Allstudies are performed with institutional ethics committee approval andpatient consent.

Study Design: This will be a Phase I, single-center, open-label,randomized, two-period crossover study in healthy male volunteers.Eighteen subjects are randomly assigned to one of two treatment sequencegroups (nine subjects/group). Relaxin is administered over two separatedosing periods as a bolus s.c. injection in the upper thigh usingequivalent doses of the PEGylated relaxin comprising a non-naturallyencoded amino acid and the commercially available product chosen. Thedose and frequency of administration of the commercially availableproduct is as instructed in the package label. Additional dosing, dosingfrequency, or other parameter as desired, using the commerciallyavailable products may be added to the study by including additionalgroups of subjects. Each dosing period is separated by a 14-day washoutperiod. Subjects are confined to the study center at least 12 hoursprior to and 72 hours following dosing for each of the two dosingperiods, but not between dosing periods. Additional groups of subjectsmay be added if there are to be additional dosing, frequency, or otherparameter, to be tested for the PEGylated relaxin as well. Theexperimental formulation of relaxin is the PEGylated relaxin comprisinga non-naturally encoded amino acid.

Blood Sampling: Serial blood is drawn by direct vein puncture before andafter administration of relaxin. Venous blood samples (5 mL) fordetermination of serum relaxin concentrations are obtained at about 30,20, and 10 minutes prior to dosing (3 baseline samples) and atapproximately the following times after dosing: 30 minutes and at 1, 2,5, 8, 12, 15, 18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample isdivided into two aliquots. All serum samples are stored at −20° C. Serumsamples are shipped on dry ice. Fasting clinical laboratory tests(hematology, serum chemistry, and urinalysis) are performed immediatelyprior to the initial dose on day 1, the morning of day 4, immediatelyprior to dosing on day 16, and the morning of day 19.

Bioanalytical Methods: An ELISA kit is used for the determination ofserum relaxin concentrations.

Safety Determinations: Vital signs are recorded immediately prior toeach dosing (Days 1 and 16), and at 6, 24, 48, and 72 hours after eachdosing. Safety determinations are based on the incidence and type ofadverse events and the changes in clinical laboratory tests frombaseline. In addition, changes from pre-study in vital signmeasurements, including blood pressure, and physical examination resultsare evaluated.

Data Analysis Post-dose serum concentration values are corrected forpre-dose baseline relaxin concentrations by subtracting from each of thepost-dose values the mean baseline relaxin concentration determined fromaveraging the relaxin levels from the three samples collected at 30, 20,and 10 minutes before dosing. Pre-dose serum relaxin concentrations arenot included in the calculation of the mean value if they are below thequantification level of the assay. Pharmacokinetic parameters aredetermined from serum concentration data corrected for baseline relaxinconcentrations. Pharmacokinetic parameters are calculated by modelindependent methods on a Digital Equipment Corporation VAX 8600 computersystem using the latest version of the BIOAVL software. The followingpharmacokinetics parameters are determined: peak serum concentration(Cmax); time to peak serum concentration (tmax); area under theconcentration-time curve (AUC) from time zero to the last blood samplingtime (AUC0-72) calculated with the use of the linear trapezoidal rule;and terminal elimination half-life (t½), computed from the eliminationrate constant. The elimination rate constant is estimated by linearregression of consecutive data points in the terminal linear region ofthe log-linear concentration-time plot. The mean, standard deviation(SD), and coefficient of variation (CV) of the pharmacokineticparameters are calculated for each treatment. The ratio of the parametermeans (preserved formulation/non-preserved formulation) is calculated.

Safety Results: The incidence of adverse events is equally distributedacross the treatment groups. There are no clinically significant changesfrom baseline or pre-study clinical laboratory tests or blood pressures,and no notable changes from pre-study in physical examination resultsand vital sign measurements. The safety profiles for the two treatmentgroups should appear similar.

Pharmacokinetic Results: Mean serum relaxin concentration-time profiles(uncorrected for baseline relaxin levels) in all 18 subjects afterreceiving PEGylated relaxin comprising a non-naturally encoded aminoacid at each time point measured. All subjects should have pre-dosebaseline relaxin concentrations within the normal physiologic range.Pharmacokinetic parameters are determined from serum data corrected forpre-dose mean baseline relaxin concentrations and the Cmax and tmax aredetermined. The mean tmax for the any clinical comparator(s) chosen issignificantly shorter than the tmax for the PEGylated relaxin comprisingthe non-naturally encoded amino acid. Terminal half-life values aresignificantly shorter for the preclinical comparator(s) tested comparedwith the terminal half-life for the PEGylated relaxin comprising anon-naturally encoded amino acid.

Although the present study is conducted in healthy male subjects,similar absorption characteristics and safety profiles would beanticipated in other patient populations: such as male or femalepatients with diabetes, male or female patients with cancer or chronicrenal failure, pediatric renal failure patients, patients in autologouspredeposit programs, or patients scheduled for elective surgery.

In conclusion, subcutaneously administered single doses of PEGylatedrelaxin comprising non-naturally encoded amino acid will be safe andwell tolerated by healthy male subjects. Based on a comparativeincidence of adverse events, clinical laboratory values, vital signs,and physical examination results, the safety profiles of thecommercially available forms of relaxin and PEGylated relaxin comprisingnon-naturally encoded amino acid will be equivalent. The PEGylatedrelaxin comprising non-naturally encoded amino acid potentially provideslarge clinical utility to patients and health care providers.

Example 39

Relaxin Functional Assay Development

This example provides the details of the relaxin functional assay.

Human peripheral blood monocytes, THP-1 cells, were used to demonstratemeasureable cAMP increase alongside positive controls Isoproterenol andForskolin. THP-1 cells were preincubated in 500 uM IBMX for 30 minutes,RLX co-stimulation with 2 uM Forskolin for 20 min. Isoproterenol,Forskolin, and relaxin polypeptides, including wild-type A (with Alaninein the 1^(st) amino acid position of the B chain, i.e., RLX-A, asopposed to RLX-D having the wild-type aspartic acid in the 1st aminoacid position of the B chain) and the variant RLX-BA1-AV13pAF (relaxinvariant with the backbone amino acid sequence of the Alanine in the1^(st) amino acid position of the B chain with a pAF substituted forposition 13 (a valine) in the A chain (i.e., RLX-A-AV13), with four (4)different size PEG's attached; 5K, 10K, 20K, and 30K. Results are shownin Table 5 for relaxin molecules attached to 20 kDa average molecularweight PEG. Also shown, among others, are results for the relaxinvariant RLX-BA1-AQ1pAF attached to a 20 kDA average molecular weight PEG(i.e., RLX-A-AQ1-20KPEG) having an alanine in the 1st amino acidposition of the B chain and a pAF substituted for position 1 (aglutamine) in the A chain.

TABLE 5 Functional Assay Raw EC50 Values [ng/mL] Sample Nov. 4, 2010Nov. 5, 2010 Nov. 5, 2010 RLX-D-WT 1.5 1.5 1.0 RLX-A-WT-001 3.6 3.3 2.6RLX-A-AQ1-20KPEG-001 38 RLX-A-AA5-20KPEG-001 41 RLX-A-AV13-20KPEG-001 56RLX-A-AR18-20KPEG-001 68 RLX-A-BV7-20KPEG-001 54 RLX-A-BA18-20KPEG-001172 RLX-A-BW28-20KPEG-001 172 RLX-A-BE5-20KPEG-001 45RLX-D-BE5-20KPEG-001 58 RLX-D-AL2-20KPEG-001 43

Example 40

This example evaluates the pharmacokinetic properties of 20 kDaPEGylated relaxin polypeptides following a single subcutaneous injectionin SD rats.

Sprague-Dawley (SD) Rats were received from Charles River Laboratories(CRL) at approximately 7-8 weeks of age (approximately 280 g at studystart). The animals were received having been jugular vein catheterizedat CRL. Animals then acclimated for 3 days prior to being placed onstudy.

Animals received a single subcutaneous injection on day 1 and PK sampleswere collected over the subsequent 80 hours. Blood samples were takenfrom animals treated with PEG-relaxin for analysis of serumconcentration according to the following sampling schedule (samplingtimes are approximate):

Day 1: pre-dose, 1, 2, 4, 8, 12, 25, 34, 50, 58, 73 and 80 hourspost-dose

Compound concentrations were measured using a bridging ECLA based on anassay which was developed at Ambrx. Concentrations were calculated usinga standard curve generated from the corresponding dosed compound andreported in an excel spreadsheet format (see appendix). Pharmacokineticparameters were estimated using the modeling program WinNonlin(Pharsight, version 5.1). Noncompartmental analysis for individualanimal data with linear-up/log-down trapezoidal integration was used,and concentration data was uniformly weighted. Compartmental analysiswas performed using two compartment. 1st order elimination model andGauss-Newton (Levenberg-Hartley) model fit equation. Table 6 shows groupmean PEG-Relaxin serum concentration values versus time. FIG. 8A-8/compares group mean serum concentration versus time for all PEG-Relaxincompounds dosed. All dose groups had measurable serum PEG-Relaxinlevels.

Individual serum concentration versus time was plotted in FIG. 8A fromanimals dosed SC with 0.5 mg/kg PEG20K-AQ1-RLX. Individual serumconcentration versus time was plotted in FIG. 9A from animals dosed SCwith 0.5 mg/kg PEG20K-AA5-RLX. Individual serum concentration versustime was plotted in FIG. 9B from animals dosed SC with 0.5 mg/kgPEG28K-AR18-RLX. Individual serum concentration versus time was plottedin FIG. 10A from animals dosed SC with 0.5 mg/kg PEG20K-BV7-RLX.Individual serum concentration versus time was plotted in FIG. 101B fromanimals dosed SC with 0.5 mg/kg PEG20K-BW28-RLX. Individual serumconcentration versus time was plotted in FIG. 1 from animals dosed SCwith 0.5 mg/kg PEG20K-AV13.

Non-compartmental analysis of serum concentration versus time data fromsubcutaneously dosed animals is summarized in Table 6.

TABLE 6 Mean serum concentrations for SD rats following a single dose ofPEG-Relaxin. Mean Dose Time Conc. SD Group Test Article (mg/kg) RouteGender (hr) (ng/mL) (ng/mL) N 1 20KPEG-AQ1  0.5 SC Male PD BQL NE 5 120KPEG-AQ1  0.5 SC Male 1 30.8 19.4 5 1 20KPEG-AQ1  0.5 SC Male 2 87.425.2 5 1 20KPEG-AQ1  0.5 SC Male 4 184.8 50.2 5 1 20KPEG-AQ1  0.5 SCMale 8 237.6 61.9 5 1 20KPEG-AQ1  0.5 SC Male 12 371.2 106.1 5 120KPEG-AQ1  0.5 SC Male 25 394.0 50.1 5 1 20KPEG-AQ1  0.5 SC Male 34278.7 59.1 5 1 20KPEG-AQ1  0.5 SC Male 50 63.4 11..9 5 1 20KPEG-AQ1  0.5SC Male 58 45.6 6.8 5 1 20KPEG-AQ1  0.5 SC Male 73 20.3 5.6 5 120KPEG-AQ1  0.5 SC Male 80 11.6 1.3 5 2 20KPEG-AA5  0.5 SC Male PD BQLNE 5 2 20KPEG-AA5  0.5 SC Male 1 19.9 6.4 5 2 20KPEG-AA5  0.5 SC Male 2100.1 51.8 5 2 20KPEG-AA5  0.5 SC Male 4 185.0 104.8 5 2 20KPEG-AA5  0.5SC Male 8 264.7 128.0 5 2 20KPEG-AA5  0.5 SC Male 12 434.3 135.0 5 220KPEG-AA5  0.5 SC Male 25 438.0 55.2 5 2 20KPEG-AA5  0.5 SC Male 34353.5 44.2 5 2 20KPEG-AA5  0.5 SC Male 50 86.9 19.1 5 2 20KPEG-AA5  0.5SC Male 58 62.4 10.6 5 2 20KPEG-AA5  0.5 SC Male 73 32.8 8.1 5 220KPEG-AA5  0.5 SC Male 80 22.1 5.4 5 3 20KPEG-AR18 0.5 SC Male PD BQLNE 5 3 20KPEG-AR18 0.5 SC Male 1 33.9 17.3 5 3 20KPEG-AR18 0.5 SC Male 2109.7 32.9 5 3 20KPEG-AR18 0.5 SC Male 4 172.7 48.2 5 3 20KPEG-AR18 0.5SC Male 8 270.5 55.3 5 3 20KPEG-AR18 0.5 SC Male 12 332.5 57.7 5 320KPEG-AR18 0.5 SC Male 25 398.6 37.6 5 3 20KPEG-AR18 0.5 SC Male 34264.3 33.8 5 3 20KPEG-AR18 0.5 SC Male 50 76.7 6.9 5 3 20KPEG-AR18 0.5SC Male 58 61.8 9.0 5 3 20KPEG-AR18 0.5 SC Male 73 25.0 4.8 5 320KPEG-AR18 0.5 SC Male 80 14.9 3.7 5 4 20KPEG-BV7  0.5 SC Male PD BQLNE 5 4 20KPEG-BV7  0.5 SC Male 1 25.7 4.6 5 4 20KPEG-BV7  0.5 SC Male 298.9 20.1 5 4 20KPEG-BV7  0.5 SC Male 4 248.5 75.5 5 4 20KPEG-BV7  0.5SC Male 8 343.5 81.8 5 4 20KPEG-BV7  0.5 SC Male 12 457.3 91.0 5 420KPEG-BV7  0.5 SC Male 25 518.5 57.7 5 4 20KPEG-BV7  0.5 SC Male 34270.4 64.5 5 4 20KPEG-BV7  0.5 SC Male 50 104.0 14.8 5 4 20KPEG-BV7  0.5SC Male 58 63.5 8.1 5 4 20KPEG-BV7  0.5 SC Male 73 26.0 3.1 5 420KPEG-BV7  0.5 SC Male 80 22.6 2.6 5 5 20KPEG-BW28 0.5 SC Male PD BQLNE 5 5 20KPEG-BW28 0.5 SC Male 1 36.4 15.4 5 5 20KPEG-BW28 0.5 SC Male 7107.9 61.5 5 5 20KPEG-BW28 0.5 SC Male 4 228.5 86.0 5 5 20KPEG-BW28 0.5SC Male 8 380.9 144.0 5 5 20KPEG-BW28 0.5 SC Male 12 486.6 135.4 5 520KPEG-BW28 0.5 SC Male 25 511.0 60.3 5 5 20KPEG-BW28 0.5 SC Male 34404.8 51.2 5 5 20KPEG-BW28 0.5 SC Male 50 184.2 31.3 5 5 20KPEG-BW28 0.5SC Male 58 122.3 37.2 5 5 20KPEG-BW28 0.5 SC Male 73 48.8 6.1 5 520KPEG-BW28 0.5 SC Male 80 37.1 5.1 5 6 20KPEG-AV13 0.5 SC Male PD BQLNE 5 6 20KPEG-AV13 0.5 SC Male 1 44.9 16.8 5 6 20KPEG-AV13 0.5 SC Male 2138.9 60.1 5 6 20KPEG-AV13 0.5 SC Male 4 345.7 117.1 5 6 20KPEG-AV13 0.5SC Male 8 533.6 157.4 5 6 20KPEG-AV13 0.5 SC Male 12 630.1 201.2 5 620KPEG-AV13 0.5 SC Male 25 742.5 117.4 5 6 20KPEG-AV13 0.5 SC Male 34540.7 31.0 5 6 20KPEG-AV13 0.5 SC Male 50 320.9 21.3 5 6 20KPEG-AV13 0.5SC Male 58 209.4 22.5 5 6 20KPEG-AV13 0.5 SC Male 73 75.0 4.8 5 620KPEG-AV13 0.5 SC Male 80 58.7 7.7 5 NE, not evaluated; BQL, belowquantifiable limit; PD, Pre-dose

TABLE 7 AQ1- AA5- AR18- BV7- BW28- AV13- RLX RLX RLX RLX RLX RLXTerminal HL (hr) 10.7 12.2 12.5 13.1 13.9 14.6 C_(max) (ng/mL) 394.0438.0 345.9 471.6 511.0 742.5 T_(max) (hr) 25.0 25.0 25.0 12.0 25.0 25.0AUC_(inf) (ng * hr/mL) 14237.9 16095.9 12985.5 17260.9 22191.2 32230.5Vz (mL/kg) 540.7 547.4 694.7 546.9 452.6 325.4 CL (mL/hr) 35.1 31.1 38.428.9 22.5 15.5 MRT (hr) 26.7 29.4 27.7 27.9 31.1 32.7

Concentration versus time curves were evaluated by, non-compartmentalanalysis (Pharsight, version 4.1). N=5 rats per group, terminal HL,terminal half-life, Cmax, maximum serum concentration measured; Tmax,time at which Cmax occurred; AUC_(inf), area under theconcentration-time curve for all serum sample/timepoints extrapolated toinfinity; Cl, apparent total serum clearance; Vz, apparent volume ofdistribution during terminal phase.

Dose solutions were measured with the ECLIA methods used for the serumconcentration measurements. Dosing solutions were diluted so as to bewithin the range of the assay. All 20KPEG-RLX dose solutions fell withinthe specified 30 percent difference from theoretical (PDT). Table 8below summarizes the results of the dose solution analyses for thisstudy.

TABLE 8 Nominal Pre-Dose in Conc. Dilution Conc. Buffer (DSA1) (ng/mL)Factor (ng/mL) % PDT RLX A-AQ1- 500000 20000 495012 −1 20K PEGRLX-A-AA5- 500000 20000 474478 −5 20K PEG RLX-A-AR18- 500000 20000432033 −14 20K PEG RLX-A-BV7- 500000 20000 377302 −25 20K PEG RLX-A-BV7-500000 20000 475452 −5 20K PEG RLX-A-AV13- 500000 20000 571645 14 20KPEG

Example 41

This example evaluates the pharmacokinetic properties of wild-type (WT)Relaxin compound following a single subcutaneous injection in SD rats.

SD Rats were received from Charles River Laboratories (CRL) atapproximately 5 weeks of age (approximately 280 g at study start). Theanimals were received having been jugular vein catheterized at CRL.Animals then acclimated for 3 days prior to being placed on study.

Animals received a single subcutaneous injection on day 1 and PK sampleswere collected over the subsequent 12 hours. Blood samples were takenfrom animals treated with WT rhRelaxin for analysis of serumconcentration according to the following sampling schedule (samplingtimes are approximate): Day 1: pre-dose, 0.33, 0.66, 1, 1.5, 2, 3, 4, 5,6, 9 and 12 hours post-dose.

Compound concentrations were measured using a bridging ECLA based on anassay which was developed at Ambrx. Concentrations were calculated usinga standard curve generated from the corresponding dosed compound andreported in an excel spreadsheet format (see appendix). Pharmacokineticparameters were estimated using the modeling program WinNonlin(Pharsight, version 5.1). Noncompartmental analysis for individualanimal data with linear-up/log-down trapezoidal integration was used,and concentration data was uniformly weighted.

Table 9 shows group mean wt rhRelaxin serum concentration values versustime. FIG. 12A compares group mean serum concentration versus time forwt rhRelaxin. All animals had measurable serum Relaxin levels.

Individual serum concentration versus time is plotted in FIG. 12B fromanimals dosed SC with 0.5 mg/kg wt Relaxin. Non-compartmental analysisof serum concentration versus time data from subcutaneously dosedanimals is summarized in Table 10. Table 11 is a summary of the dosesolution analyses. The dosing solutions met the acceptable criteria ofless than or equal to 30% PDT.

TABLE 9 Mean Dose Time Conc. SD Group Test Article (mg/kg) Route Gender(hr) (ng/mL) (ng/mL) N 1 wt rhRelaxin 0.5 SC Male PD BQL NE 5 1 wtrhRelaxin 0.5 SC Male 0.33 244.0 18.7 5 1 wt rhRelaxin 0.5 SC Male 0.66227.9 45.6 5 1 wt rhRelaxin 0.5 SC Male 1 211.7 45.9 5 1 wt rhRelaxin0.5 SC Male 1.5 166.7 38.7 5 1 wt rhRelaxin 0.5 SC Male 2 119.9 24.5 5 1wt rhRelaxin 0.5 SC Male 3 52.5 23.0 S 1 wt rhRelaxin 0.5 SC Male 4 24.111.4 5 1 wt rhRelaxin 0.5 SC Male 5 7.7 2.6 5 1 wt rhRelaxin 0.5 SC Male6 BQL NE 5 1 wt rhRelaxin 0.5 SC Male 9 BQL NE 5 1 wt rhRelaxin 0.5 SCMale 12 BQL NE 5 NE, not evaluated; BQL, below quantifiable limit; PD,Pre-dose

TABLE 10 Pharmacokinetic parameter values for wt rhRelaxin dosed in SDrats. wt rh Relaxin Terminal HL 0.8 (0.1) (hr) C_(max) (ng/mL) 258.1(26.1)  T_(max) (hr) 0.5 (0.2) AUC_(inf) 508.9 (81.8)  (ng * hr/mL) Vz(mL/kg) 1159 (284)  CL (mL/hr) 1006 (185)  MRT (hr) 1.56 (0.16)

Concentration versus time curves were evaluated by non-compartmentalanalysis (Pharsight, version 4.1). N=5 rats per group, terminal HL,terminal half-life; Cmax, maximum serum concentration measured; Tmax,time at which Cmax occurred: AUC_(inf), area under theconcentration-time curve for all serum sample/timepoints extrapolated toinfinity; Cl, apparent total serum clearance; Vz, apparent volume ofdistribution during terminal phase. Numbers are mean with SD inparentheses.

Table 11

Dose solutions were measured with the ECLA methods used for the serumconcentration measurements. Dosing solutions were diluted so as to bewithin the range of the assay. All wt rhRelaxin dose solutions fellwithin the specified 30 percent difference from theoretical (PDT). Table11 below summarizes the results of the dose solution analyses for thisstudy.

TABLE 11 Dose solution analyses of test article. Nominal Conc. DilutionConc. % Dose Solution Analysis (ng/mL) Factor (ng/mL) PDT 0.5 mg/mLPre-Dose in 250000 10000 208830 −16 formulation buffer (DSA1) 0.5 mg/mLPre-Dose in 250000 10000 225898 −10 serum (DSA2)

Example 42

This example evaluated the pharmacokinetic properties of a 20 kDaPEGylated Relaxin compound, RLX-A-AQ1-20KPEG, following a singlesubcutaneous or intravenous injection in SD rats.

SD Rats were received from Charles River Laboratories (CRL) atapproximately 7-8 weeks of age (approximately 280 g at study start). Theanimals were received having been jugular vein catheterized at CRL.Animals then acclimated for 3 days prior to being placed on study.Animals received a single subcutaneous injection on day 1 and PK sampleswere collected over the subsequent 82 hours. Blood samples were takenfrom animals treated with PEG-Relaxin for analysis of serumconcentration according to the following sampling schedule (samplingtimes are approximate): Day 1: pre-dose, 1, 3, 5, 10, 25, 34, 48, 58, 72and 82 hours post-dose.

Compound concentrations were measured using a bridging ECLA based on anassay which was developed at Ambrx. Concentrations were calculated usinga standard curve generated from the corresponding dosed compound andreported in an excel spreadsheet format (see appendix). Pharmacokineticparameters were estimated using the modeling program WinNonlin(Pharsight, version 5.1). Noncompartmental analysis for individualanimal data with linear-up/log-down trapezoidal integration was used,and concentration data was uniformly weighted. Compartmental analysiswas performed using two compartment. 1st order elimination model andGauss-Newton (Levenberg-Hartley) model fit equation.

FIG. 13A shows a comparison of group mean serum concentration versustime for all PEG-RLX groups dosed subcutaneously or intravenously. Asingle dose injection was administered to each animal. N=3-5 animals pergroup.

FIG. 13B shows a graph of individual animal serum concentration timecurves for SD rats dosed intravenously with 0.25 mg/kg of20KPEG-AQ1-RLX. A single, intravenous dose was administered to eachanimal. N=4 animals per group.

FIG. 14A shows a graph of individual animal serum concentration timecurves for SD rats dosed subcutaneously with 0.5 mg/kg of PEG20-AQ1-RLX.A single, subcutaneous dose was administered to each animal. N=5 animalsper group.

FIG. 14B shows a graph of individual animal serum concentration timecurves for SD rats dosed subcutaneously with 0.25 mg/kg ofPEG20-AQ1-RLX. A single, subcutaneous dose was administered to eachanimal. N=3 animals per group.

FIG. 15 shows a graph of individual animal serum concentration timecurves for SD rats dosed intravenously with 0.125 mg/kg ofPEG20-AQ1-RLX. A single, intravenous dose was administered to eachanimal. N=5 animals per group.

FIG. 16A shows a graph of the mean PEG-Relaxin Terminal Half-life versusdose; error bars=SD. FIG. 16B shows a graph of mean PEG-RelaxinAUC_(inf) versus dose; error bars=SD.

FIG. 17A shows a graph of the mean PEG-Relaxin Cmax versus dose; errorbar=SD.

FIG. 17B shows a graph of the mean PEG-Relaxin Clearance versus dose;error bars=SD.

FIG. 18A shows a graph of the mean PEG-Relaxin Volume of distributionversus Dose; error bars=SD. FIG. 18B shows a graph of a comparison ofSerum-time concentration after a single intravenous or subcutaneousinjection of 0.25 mg/kg of 20KPEG-AQ1 Relaxin.

Table 12 shows group mean PEG-Relaxin serum concentration values versustime.

TABLE 12 Mean Dose Time Conc. SD Group Test Article (mg/kg) Route Gender(hr) (ng/mL) (ng/mL) N 1 20KPEG-AQ1  0.25 IV Male PD BQL NE 4 120KPEG-AQ1  0.25 IV Male 1 2912.8 203.7 4 1 20KPEG-AQ1  0.25 IV Male 31310.8 115.2 4 1 20KPEG-AQ1  0.25 IV Male 5 700.7 68.3 4 1 20KPEG-AQ1 0.25 IV Male 10 241.6 27.2 4 1 20KPEG-AQ1  0.25 IV Male 25 61.2 5.4 4 120KPEG-AQ1  0.25 IV Male 34 27.0 0.6 4 1 20KPEG-AQ1  0.25 IV Male 4813.7 2.2 4 1 20KPEG-AQ1  0.25 IV Male 58 8.0 2.2 4 1 20KPEG-AQ1  0.25 IVMale 72 4.3 1.4 4 1 20KPEG-AQ1  0.25 IV Male 82 2.2 0.6 4 2 20KPEG-AQ10.5 SC Male PD BQL NE 5 2 20KPEG-AQ1 0.5 SC Male 1 16.3 4.4 5 220KPEG-AQ1 0.5 SC Male 3 75.8 20.2 5 2 20KPEG-AQ1 0.5 SC Male 5 96.326.7 5 2 20KPEG-AQ1 0.5 SC Male 10 135.1 30.1 5 2 20KPEG-AQ1 0.5 SC Male25 257.4 48.5 5 2 20KPEG-AQ1 0.5 SC Male 34 184.9 26.8 5 2 20KPEG-AQ10.5 SC Male 48 159.9 31.7 5 2 20KPEG-AQ1 0.5 SC Male 58 86.5 24.6 5 220KPEG-AQ1 0.5 SC Male 72 20.3 1.4 5 2 20KPEG-AQ1 0.5 SC Male 82 11.81.1 5 3 20KPEG-AQ1  0.25 SC Male PD BQL NE 3 3 20KPEG-AQ1  0.25 SC Male1 12.8 1.3 3 3 20KPEG-AQ1  0.25 SC Male 3 45.3 6.9 3 3 20KPEG-AQ1  0.25SC Male 5 62.2 8.6 3 3 20KPEG-AQ1  0.25 SC Male 10 90.1 10.9 3 320KPEG-AQ1  0.25 SC Male 25 127.4 20.2 3 3 20KPEG-AQ1  0.25 SC Male 3483.2 13.8 3 3 20KPEG-AQ1  0.25 SC Male 48 32.6 2.6 3 3 20KPEG-AQ1  0.25SC Male 58 16.3 0.3 3 3 20KPEG-AQ1  0.25 SC Male 72 4.9 0.9 3 320KPEG-AQ1  0.25 SC Male 82 2.7 0.4 3 4 20KPEG-AQ1  0.125 SC Male PD BQLNE 5 4 20KPEG-AQ1  0.125 SC Male 1 5.7 1.5 5 4 20KPEG-AQ1  0.125 SC Male3 26.4 6.2 5 4 20KPEG-AQ1  0.125 SC Male 5 37.2 8.6 5 4 20KPEG-AQ1 0.125 SC Male 10 50.1 6.1 5 4 20KPEG-AQ1  0.125 SC Male 25 75.9 8.8 5 420KPEG-AQ1  0.125 SC Male 34 46.9 6.7 5 4 20KPEG-AQ1  0.125 SC Male 4820.8 8.2 5 4 20KPEG-AQ1  0.125 SC Male 58 8.4 2.4 5 4 20KPEG-AQ1  0.125SC Male 72 2.3 0.8 5 4 20KPEG-AQ1  0.125 SC Male 82 1.3 0.4 5 NE, notevaluated; BQL, below quantifiable limit; PD, Pre-dose

Example 43

This example evaluated the pharmacologically active dose and systemicexposure of wild-type relaxin and a PEG-relaxin variant,RLX-A-AQ1-20KPEG, in female Long-Evans rats.

In human pregnancy, blood volume increases greatly and plasma osmolalityis reduced due to mild hyponatremia. Based on studies in rodents, theeffects of relaxin on the osmotic threshold during pregnancy areprobably mediated, at least in part, through its effects on drinking andvasopressin secretion from the posterior pituitary. Specific bindingsites for relaxin occur in two of the rat forebrain circumventricularorgans, the organum vasculosum of the Lamina terminalis and subfornicalorgan which connect into the hypothalamic/pituitary axis. Innon-pregnant rats, when human relaxin (WT-RLX) was administered viaosmotic pump to generate a blood level similar to rat pregnancy levelsat around 2 weeks post-conception (˜10-15 ng/mL), plasma osmolalitydecreased by 3-4 percent (Danielson and Conrad, J Appl Physiol, 2003.95(4): p. 1509-14). In addition to effects related to changes in osmoticthreshold, it is also possible that increased natriuresis could beplaying a contributory role. Using a rat model similar to that describedabove, RLX-A-AQ1-20KPEG was investigated for effects on plasma sodiumconcentration and osmolality.

The objective of these signal generation studies was to establish invivo activity and define a pharmacologically active dose of thePEG-relaxin variant. To accomplish these objectives, physiologicallyrelevant endpoints responsive to relaxin were evaluated in femaleLong-Evans rats including water intake, urine output, and select urineand blood clinical chemistry. To enable identification of candidateendpoints, wild-type relaxin was tested first (Phase 1; results depictedin FIG. 19A-19F), at doses calculated to achieve plasma concentrationsof 0.3×, 1× and 10× of an in vitro target inhibition. Target doses weredelivered by bolus intravenous (TV) dosing followed by 6-hour continuousinfusion. Doses of 0.3×, 1×, and 10× wild-type relaxin induced increasesof 95%, 68%, and 32%, respectively, in water intake compared to vehicle.Changes in mean plasma sodium concentrations from baseline to 2 and 6hours were −9.5 and 1.2 mEq/L for vehicle (0×); −5.8 and −9.0 mEq/L at0.3×; 1.0 and −1.5 mEq/L at 1×; −4.7 and −1.4 mEq/L at 10×. The changesin plasma osmolarity from baseline to 2 and 6 hours were −20.7 and 1.0mosmol/kg water for vehicle (0×); −11.0 and −19.1 mosmol/kg water at0.3×; 0.3 and −4.4 mosmol/kg water for 1×; −10.3 and −3.8 mosmol/kgwater for 10×.

PEG-relaxin, specifically RLX-A-AQ1-20KPEG. (Phase 2; results depictedin FIG. 20A-20I) was administered at a bolus volume of 1 μL per gram ofbody weight with blood collected 2 and 6 hours after dosing and urinewas collected for the 6 hour period following dosing. Treatment withPEG-relaxin at 0.1×, 0.3× and 1× doses resulted in 93%, 128% and 105%increases, respectively, in water intake compared to the vehicle group.Changes in mean plasma sodium concentration from baseline to 2 and 6hours were 2.5 and 1.5 mEq/L for vehicle (0×); −1.5 and 4.8 mEq/L at0.1×; −4.0 and −2.9 mEq/L at 0.3×; −2.3 and −4.0 mEq/L at 1×. Changes inplasma osmolarity from baseline to 2 and 6 hours were 4.5 and 2.1mosmol/kg water for vehicle (0×); −5.1 and −11.0 mosmol/kg water at0.1×; −8.2 and −5.0 mosmol/kg water at 0.3×; −5.6 and −9.5 mosmol/kgwater at 1×. There were no clear changes in urine clinical chemistryvalues following treatment with wild-type or PEG-relaxin. These dataestablish in vivo activity and enable rationale dose selection forsubsequent in vivo disease model studies with PEG-relaxin.

TABLE 13 Animal Group Design Plasma Concentration IV Bolus Dose IVInfusion Animal (Fold Increase Concentration Concentration # AnimalsGroup Test Article IDs Over Target) (mg/mL) (mg/mL)^(c) per Group Phase1 Wild-Type Relaxin Dosing^(a) 1  Wild type- 1F001-   1X  0.003  0.08 4relaxin 1F004  2  Wild type- 2F001-  10X  0.03 0.8 4 relaxin 2F004 3^(b) Wild type- 3F001- vehicle vehicle vehicle 4 relaxin 3F004  4^(b)Wild type- 4F001- 0.3X  0.001  0.026 4 relaxin 4F004  ^(a)Groups 1 and 2(1x and 10x) were evaluated first, and because test article-relatedeffects on drinking water consumption, hematocrit, and urine output wereapparent, doses for the second set of rats were revised to vehicle and0.3X in Groups 3 and 4, respectively. There was a washout period of atleast 36 hours between the Groups 1 and 2. A washout period of at least36 hours also occurred between Groups 3 and 4. ^(b)Rats in Group 1 weresubjected to a second randomization and assigned to Group 2 followingthe washout period. Rats in Group 3 were subjected to a secondrandomization and assigned to Group 4. Animal numbers, 001-004 remainedthe same in Groups 2 and 4 as they were in Groups 1 and 3; however, thegroup number designations, Group 2 and Group 4, were used to enable tubelabeling and sample identification clarity. ^(c)Wild-type relaxin wasinjected intravenously in a bolus volume of 100 μL followed by aninfusion rate of 50 μL/hour.

TABLE 14 Plasma IV Bolus Concen- Dose # tration Concen- Animals TestAnimal (Fold Increase tration per Group Article IDs Over Target)(mg/mL)^(a) Group Phase 2 PEG-Relaxin Dosing 5 PEG- 5F001- Vehicle NA 4Relaxin 5F004 6 PEG- 6F001- 0.3X 0.03 4 Relaxin 6F004 7 PEG- 7F001- 0.1X0.01 4 Relaxin 7F004 8 PEG- 8F001- 1.0X 0.1 4 Relaxin 8E004 ^(a)Bolusdose was administered at a volume of 1 μL/g body weight.

Phase 1 Wild-Type Relaxin

Female Long-Evans rats 12-14 weeks of age with bi-lateral jugular veincatheters surgically placed by the vendor were obtained from CharlesRiver Laboratories. Two sets of four rats were used. Each set was usedto evaluate two doses of wild-type relaxin. There was a washout periodof 36 hours or more between Groups 1 and 2 and Groups 3 and 4 as ratsfrom Groups 1 and 3 were reused in Groups 2 and 4, respectively. Ratshad ad libitum access to food and water and were housed in the Culex ABSmetabolic caging system (Culex Automated Blood Sampler. BioanalyticalSystem Inc) the night prior to the initiation of dosing and for the 6hours after the IV bolus dose and initiation of infusion dosing.

Phase 1 Wiki-Type Relaxin Dose Administration

In phase 1 each rat was given a one-time IV bolus and a 6 hour IVinfusion. The IV bolus injection via surgically implanted jugularcatheters followed by IV infusion via surgically implanted jugularcatheters. At least 36 hours between the Groups 1 and 2 in the first setof rats and Groups 3 and 4 in the second set of rats was allows forwashout.

Phase 2 PEG-Relaxin

Female Long-Evans rats 12-14 weeks of age with bi-lateral jugular veincatheters surgically placed by the vendor were obtained from CharlesRiver Laboratories. Four sets of four rats were used, with each set usedto evaluate one dose of PEG-Relaxin. Rats receiving PEG-Relaxin were notreused following a washout period due to the extended plasma exposure ofPEGylated compounds. Rats had ad libitum access to food and water andwere housed in the Culex ABS metabolic cages.

Phase 2 PEG-Relaxin Dose Administration

In Phase 2, the rats received a one-time IV injection via surgicallyimplanted jugular catheters.

All bolus and IV dosing solutions were formulated according to thesethawing and mixing instructions for all dose solutions:

The frozen sample bottle for each dosing formulation was allowed to thawat 4° C. for 3 to 4 hours, with hourly inspections of the thawingprocess. Once thawing was complete, the formulation was mixed with agentle inversion taking care not to create bubbles. Care was taken togently and thoroughly mix each formulation just prior to intravenousdosing. Each dosing formulation was maintained at 4° C. until used fordosing.

The doses were prepared immediately prior to preparation and each groupreceived dose concentrations of the following:

Phase 1 (Groups 1-4) Group 1: (1X) IV Bolus 0.003 mg/mL IV Infusion 0.08mg/mL Group 2: (10X) IV Bolus 0.03 mg/mL IV Infusion 0.8 mg/mL Group 3:(vehicle) Bolus and infusion Group 4: (0.3X) IV Bolus 0.001 mg/mL IVInfusion 0.026 mg/mL Phase 2 (Groups 5-8) Group 5: Vehicle IV Bolus NAGroup 6: (0.3X) IV Bolus 0.03 mg/mL Group 7: (0.1X) IV Bolus 0.01 mg/mLGroup 8: (1.0X) IV Bolus 0.1 mg/mL

Animals used on this study were selected on the basis of acceptablefindings from body weight measurements, jugular catheter patency, andfunctionality. Animals identified with catheters unsuitable for dosingor blood collection prior to study start or during the blood collectionperiod following initiation of dosing were removed from the study andreplaced by rats with suitable catheters. Replacement of catheterizedrats and compound dosing occurred at the earliest time possible relativeto the dosing schedule, staffing availability, and receipt of rats fromthe vendor.

Phase 1: The animals were randomized according to pre-study body weightand assigned to Group 1. Following completion of dosing, rats in Group 1were subjected to a second randomization following the washout periodand reassigned to Group 2. Following completion of dosing, rats in Group3 were subjected to a second randomization following the washout periodand reassigned to Group 4.

Phase 2: The animals were randomized according to pre-study body weightand assigned to Groups 5 through 8.

Daily at approximately 30 minutes and 1, 2, 4, and 6 hours afterinitiation of dosing. Visual inspections of physical and behavioralchanges were performed and animals were examined for changes in bodyposture, hair coat, activity, excreta, etc. Routine body weights weretaken and recorded prior to dosing.

Blood and Urine Sample, Collection, Handling, and Analysis

Phase 1 Study Protocol

Baseline urine collection were started the night prior to doseadministrations, continued for a period of 15-18 hours, and werecollected into a chilled (wet ice) vial. Baseline blood samples (˜400 μLwhole blood) were collected at the end of baseline urine collection. Onthe day of the experiment, rats were given an IV bolus injection of thetest article followed by continuous infusion with wild-type relaxin at aconstant infusion rate delivered by a syringe pump (Harvard 11 plus) for6 hours via left jugular vein catheter at volumes of 50 μL/hour. Twoadditional blood samples (˜˜400 μL whole blood each) were collected at 2and 6 hours post-relaxin infusion. All blood samples were collected viaright jugular catheter into sample tubes containing K3EDTA anticoagulantusing Culex ABS programmed sampling method and samples were stored in arefrigerated environment until sample processing. Urine was continuouslycollected into a chilled vial throughout the duration of the relaxininfusion period. The total urine volume from each collection wasrecorded, and 2-5 mL of each urine sample was stored in a Seventh WaveLabs freezer set to maintain approximately −80°. Water intake during the6-hour infusion period was recorded by comparing pre- and post-infusionweight of bottle+water. The infusion pump was stopped after 6 hours ofcontinuous infusion, and rats underwent a washout period of at least 36hours before they were subjected to the second dose evaluation.

Continuous IV infusion was required to maintain targeted, stable plasmadrug concentration.

Phase 2 Study Protocol

Baseline urine collection started the night prior to doseadministrations for a period of 15-18 hours, and samples were collectedinto a chilled vial. Baseline blood samples (˜400 μL whole blood) werecollected at the end of the baseline urine collection period. On the dayof the experiment, rats were administered a single dose of PEG-Relaxinintravenously at a volume not to exceed 10 mL/kg/day (one time dose).Two additional blood samples (˜400 μL whole blood) were collected at 2and 6 hours post-relaxin administration. All blood samples werecollected via right jugular catheter into sample tubes containing K3EDTAanticoagulant using Culex ABS programmed sampling method and stored in arefrigerated environment until processing. Urine samples werecontinuously collected from time 0 to 6 hours post administration ofPEG-Relaxin into a chilled vial during sampling. The total urine volumefrom each collection was recorded, and 2-5 mL of each urine sample wasstored in a Seventh Wave Labs freezer set to maintain approximately −80°C. Rats were euthanized 6 hours after dosing. Water intake during the6-hour infusion period was recorded by comparing weight of thebottle+water at the beginning and the end of drug infusion period.

Pathology

Urine samples were collected as already described in this example. Twoto 5 mL of chilled urine samples were frozen on dry ice and stored in afreezer set to maintain approximately −80° C. until shipped on dry iceto AVL for analysis of urine creatinine and BUN for determination ofcreatinine clearance.

A blood sample was collected using the Culex ABS using K₃EDTA as ananticoagulant. Fifteen microliters of whole blood samples were filledinto untreated capillary tubes, sealed with clay on one end, andcentrifuged in a hematocrit centrifuge (International Equipment Company,IEC MB centrifuge) for five minutes. Hematocrit results were obtainedusing a microhematocrit reading device provided by the manufacturer.

Blood was collected for potential determination of systemic exposure ofwild-type and PEG-Relaxin in accordance with the collection schedule andprocedures listed below. There were three collection intervals.Collection time points were 0, 2, and 6 hours post-dose for Phase 1 andPhase 2 rats. Phase 1: 8 animals per time point. Phase 2: 16 animals pertime point. Collection volume was nearly 400 micro liters of wholeblood. All animals in Groups 1 through 8 were bled at three time points:baseline (pre-dose t=0) and 2 and 6 hours post-initiation of infusionfor wild-type relaxin IV dosing and post IV dosing for PEG-Relaxindosing groups, respectively. The time of initiation of IV infusiondosing and the actual time of each bleed were recorded in the raw datafor each animal. Per Sponsor decision, blood was not sent to Ambrx forsystemic exposure determinations during the study conduct. Samples havebeen stored frozen at in a Seventh Wave Labs freezer set to maintainapproximately −80° C., and will be returned to Sponsor.

Phase 1 Wildtype Relaxin RESULTS

FIG. 19A-19F shows data from Phase 1. FIG. 19A shows the effect of IVinfusion with wild-type relaxin on water intake and urine output. FIG.19B shows a baseline for each group of Long-Evans rats from −16 ours to0 of Phase 1 for urine output. FIG. 19C shows the effect of IV infusionwith wild-type relaxin on hematocrit. FIG. 19D shows the effect of IVinfusion with wild-type relaxin on plasma BUN in the female Long-Evansrats. FIG. 19E shows water intake for each group of Long-Evans rats from0 to 6 hours of Phase 1. FIG. 19F shows urine output for each group ofLong-Evans rats from 0 to 6 hours of Phase 1.

The average water intake during the 6 hour infusion in vehicle (0×)treated rats was 1.8 mL per 100 grams of body weight, which increased to3.6, 3.1, and 2.4 mL per 100 grams of body weight in the 0.3×, 1× and10× wild-type relaxin treated rats, respectively. These are 95%, 68%,32% increases over vehicle for the 0.3×, 1× and 10× dosing groupsrespectively.

Average hematocrit at baseline, 2 and 6 hours after initiation of dosingwas 35.1%, 35.0%, and 34.3% in vehicle treated rats, respectively;33.1%, 33.7%, and 32.3% in the 0.3× group; 31.4T, 32.0% and 33.7% in the1× group; 29.7%, 31.9% and 30.4% in the 10× group.

The average urine output during the 6 hour infusion was 1.8, 1.5, 1.2and 1.2 mL per 100 grams of body weight for the vehicle (0×), 0.3×, 1×and 10× groups respectively.

The mean plasma sodium concentration at baseline, 2 and 6 hours afterinitiation of dosing was 139.3, 129.8, 140.5 mEq/L in vehicle (0×)group; 141.8, 136.0 and 132.8 mEq/L in the 0.3× group; 138.8, 139.8 and137.3 mEq/L in 1× group; 137.0, 132.3 and 135.6 mEq/L in 10× group. Thechange from baseline at 2 and 6 hours was −9.5 and 1.2 mEq/L for vehicle(0×); −5.8 and −9.5 mEq/L at 0.3×; 1.0 and −1.5 mEq/L at 1×; −4.7 and−1.4 mEq/L at 10×.

Plasma osmolarity was calculated using the following equation:osmolarity (OSM)=(2*na)+(Glu/18)+(BUN/2.8). The mean plasma osmolarityat baseline, 2 and 6 hours after initiation of 277.3 mosmol/kg water inthe 0.3× group; 291.0, 291.3 and 286.6 mosmol/kg water in the 1× group;286.4, 276.1 and 282.6 mosmol/kg water in the 10× group. The change frombaseline at 2 and 6 hours was −10.7 and 1.0 mosmol/kg water for vehicle(0×); =11.0 and =19.1 mosmol/kg water at 0.3×; 0.3 and −4.4 mosmol/kgwater for 1×; −10.3 and −3.8 mosmol/kg water for 10×. Urine clinicalchemistry data of BUN/Cr, Na excretion are summarized in the tablesbelow:

TABLE 15 Urine Clinical Chemistry Data Summary of Wild-Type Relaxin(Phase 1 Study) Na BUN Cr Cl BUN/ Excretion Excretion (mL/min/ Groups Crsem (mEq/hr) sem (mg/hr) sem 100 g) sem Vehicle (baseline) 36 12 0.0830.010 13.5  4.4 0.65 0.04 Vehicle (6 hr study) 26  3 0.079 0.018 9.4 2.30.60 0.15 0.3X (baseline) 66  9 0.055 0.005 17.3  3.9 0.38 0.06 0.3X (6hr study) 43  6 0.071 0.015 12.1  1.3 0.45 0.07 1X (baseline) 18  20.046 0.010 7.6 1.5 0.62 0.09 1X (6 hr study) 19  4 0.030 0.003 4.6 1.50.36 0.05 10X (baseline) 21  2 0.063 0.001 8.5 1.1 0.69 0.04 10X (6 hrstudy) 17  4 0.055 0.005 4.9 0.7 0.81 0.11

TABLE 16 Urine Clinical Chemistry Raw Data of Wild-Type Relaxin (Phase 1Study) CrCl DOSE Group ID K Na Great BUN (ml/min/100 g) Vehicle 3F001 T068232 136 119 84 1080 0.702 Vehicle 3F001 T6 68233 74 67 33 850 0.257Vehicle 3F002 T0 68234 122 130 59 980 0.692 Vehicle 3F002 T6 68235 99 6639 920 0.979 Vehicle 3F003 T0 68236 276 130 54 2950 0.694 Vehicle 3F003T6 68237 110 154 58 1980 0.552 Vehicle 3F004 T0 68238 355 126 52 30000.522 Vehicle 3F004 T6 68239 176 152 71 1320 0.614 0.3X 4F001 T0 68791272 47 22 1950 0.413 0.3X 4F001 T6 68792 174 112 25 1500 0.356 0.3X4F002 T0 68793 274 68 42 1860 0.522 0,3X 4F002 T6 68794 180 88 42 15000.657 0.3X 4F003 T0 68795 230 54 25 1800 0.329 0.3X 4F003 T6 68796 18082 60 1880 0.449 0.3X 4F004 T0 68797 225 68 23 1440 0.260 0.3X 4F004 T668798 365 152 68 3100 0.352   1X 1F001-T0 67488 194 <40 64 1480 0.526  1X 1F001-T6 67489 138 62 57 960 0.394   1X 1F002-T0 67487 203 74 931540 0.579   1X 1F002-T6 67496 296 161 88 1300 0.337   1X 1F003-T0 67490110 85 64 820 0.495   1X 1F003-T6 67495 176 167 104 1440 0.236   1X1F004-T0 67483 224 74 67 1420 0.882   1X 1F004-T6 67492 94 <40 24 7400.476  10X 2F001-T0 67497 140 64 40 900 0.670  10X 2F001-T6 67498 54 6864 500 0.729  10X 2F002-T0 67484 166 101 75 1120 0.598  10X 2F002-T667485 214 126 57 1260 0.618  10X 2F003-T0 67491 254 <40 103 1650 0.696 10X 2F003-T6 67486 60 <40 106 800 1.129  10X 2F004-T0 67494 294 88 741600 0.809  10X 2F004-T6 67493 212 125 85 1400 0.756 Units: Na (mEq/L);Creatinine (mg/dL); BUN (mg/dL), glucose (mg/dL), K (mmol/L), CrCl:mL/min/100 g BW Creatinine Clearance (CrCl) = (U creat. * U vol)/(Pcreat. * U time)

TABLE 17 Physiology Data Summary of Wild-Type Relaxin (Phase 1 Study)Water Intake Baseline Urine Urine (0-6 hr) (−16-0 hr) (0-6 hr) mL/100 gBW mL/100 g BW mL/100 g BW Dose mean sem mean sem mean sem Vehicle 1.80.4 4.1 0.4 1.8 0.3 0.3X 3.6 0.2 5.7 1.1 1.5 0.4   1X 3.1 0.6 3.8 0.61.2 0.6  10X 2.4 0.7 4.3 0.8 1.2 0.1 Hematocrit (%) 0 hr 2 hr 6 hr Dosemean sem mean sem mean sem Vehicle 35.1  1.0 35.0  0.9 34.3  0.5 0.3X29.7  1.1 31.9  1.2 30.4  0.4   1X 31.4  2.7 32.0  2.9 33.7  2.1  10X29.7  1.1 31.9  1.2 30.4  0.4

Phase 2 PEG-Relaxin Variant RESULTS

FIG. 20A-20I shows data from Phase 2 following administration of vehicle(control group) and test groups with 0.1×, 0.3× and 1× administration ofa 20K PEG-Relaxin variant with A chain substitution in position 1 withpAF bound to PEG (RLX-A-AQ1-20KPEG). FIG. 20A shows the effect of onwater intake and urine output. FIG. 20B shows the effect on plasmasodium levels for each group of Long-Evans rats following injection.FIG. 20C shows the effect on plasma sodium change levels for each groupof Long-Evans rats following injection. FIG. 20D shows the effect ofPEG-Relaxin on plasma osmolarity. FIG. 20E shows the effect of IVinfusion with PEG-Relaxin on plasma osmolarity change. FIG. 20F showsthe effect of PEG-Relaxin administration on BUN levels. FIG. 20G showsthe effect of PEG-relaxin administration on water intake for each groupof Long-Evans rats from 0 to 6 hours of Phase 2. FIG. 20H shows baselineurine output for each group of Long-Evans rats from −16 to 0 hours ofPhase 2. FIG. 20I shows the effect of PEG-relaxin administration onurine output for each group of Long-Evans rats from 0 to 6 hours ofPhase 2.

The average water intake during the 6 hour post-dose period in vehicle(0×) treated rats was 1.5 mL per 100 grams of body weight, whichincreased to 2.9, 3.4, and 3.1 mL per 100 grams of body weight in 0.1×,0.3× and 1×PEG-relaxin variant treated rats, respectively. Theserepresent 93%, 128% and 105% increases over the mean of vehicle groupsfor the 0.1×, 0.3× and 1× groups respectively.

The average urine output during the 6 hour post-dose period was 0.9,0.2, 0.5 and 0.4 mL per 100 grams of body weight for vehicle (0×, 0.3×and 1× groups respectively). The mean plasma sodium concentrations atbaseline, 2 and 6 hours after dosing were 136.3, 138.8 and 137.8 mEq/Lin the vehicle (0×) group, respectively; 138.5, 137.0 and 133.7 mEq/L inthe 0.1× group, respectively; 137.5, 133.5 and 134.6 mEq/L in the 0.3×group, respectively; 137.3, 135.0 and 133.3 mEq/L in the 1× group,respectively. The change from baseline at 2 and 6 hours was 2.5 and 1.5mEq/L for vehicle (0×); −1.5 and −4.8 mEq/L at 0.1×; −4.0 and −2.9 mEq/Lat 0.3×; −2.3 and −4.0 mEq/L at 1×.

The mean plasma osmolarity at baseline, 2 and 6 hours after dosing was285.4, 289.9, and 287.5 mosmol/kg water in the vehicle (0×) group,respectively; 292.7, 287.6 and 281.7 mosmol/kg water in the 0.1× group,respectively; 287.6, 279.4 and 282.6 mosmol/kg water in the 0.3× group,respectively; 289.3, 283.7 and 279.4 mosmol/kg water in the 1× group,respectively. The change from baseline at 2 and 6 hours was 4.5 and 2.1mosmol/kg water for vehicle (0×); −5.1 and −11.0 mosmol/kg water at0.1×; −8.2 and −5.0 mosmol/kg water for 0.3×; −5.6 and −9.9 mosmol kgwater for 1×.

In summary, both plasma sodium concentration and osmolality decreasedwith time in response to IV bolus administration of RLX-A-AQ1-20KPEG,with both endpoints essentially mirroring each other with respect tomagnitude of response. A clear downward trend was observed for all threeRLX-A-AQ1-20KPEG treatment groups, with several data points reachingstatistical significance (P<0.05) when compared to vehicle treatedanimals. For all RLX-A-AQ1-20KPEG treatment groups, when compared tobaseline values, plasma sodium and Posmol both decreased by ˜2-4%. Nosignificant changes over time were observed for vehicle treated animals.With the single exception of BUN levels being slightly lower at the 6 hrtime point for Group 4 (P=0.024), plasma glucose and BUN levels were notstatistically different when drug-treated animals (Groups 2-4) werecompared either to Group 1 (vehicle) or to each other (data not shown).These data provide evidence that the observed mild hyponatremia waslargely responsible for the changes in Posmol. Decreases in plasmasodium and Posmol were greater for Group 3 (mid-dose) and Group 4(high-dose) at the 2 hr time point when compared to Group 2 (low-dose).However, at the 6 hr time point all three doses of RLX-A-AQ1-20KPEGgenerated essentially equivalent decreases with no obvious evidence of adose-response.

Example 44

In Vitro Activation of Relaxin Receptors by PEG-Relaxin

This example further characterizes the in vitro activity of a modifiedrelaxin, specifically RLX-A-AQ1-20KPEG, as compared to unmodifiedwild-type relaxin (RLX-WT). The in vitro assays utilized multiple celltypes including THP-1 monocytic cells, CHO-K1 cells stably expressingRXFP receptors.

Relaxin cellular signaling mechanisms are quite diverse, cell-typedependent and have been the subject of many reviews. One of the mostextensively studied cell signaling mechanisms is relaxin's ability torapidly (1-5 minutes) increase intracellular levels of cAMP throughG_(αs) stimulation of adenylate cyclase (Anand-Ivell, R., et al., MolCell Endocrinol, 2007. 272(1-2): p. 1-13). Rapid stimulation of cAMP hasbeen demonstrated for relaxin in THP-1 human monocytic cells whichendogenously expresses the RXFP1 receptor (id.) as well as in stablytransfected HEK-293T cells expressing human RXFP1 (Halls et al., MolPharmacol, 2006. 70(1): p. 214-26). Increases in intracellular cAMP inresponse to relaxin treatment have also been reported for primarycultures of rat renal and cardiac fibroblasts (Halls et al., Ann N YAcad Sci, 2009. 1160: p. 108-11), albeit the level of cAMP induction wasmuch less robust as compared to RXFP1 HEK-293T cells. To furthercharacterize their relative potency and intrinsic activity.RLX-A-AQ1-20KPEG and WT-RLX were tested head-to-head for their abilityto increase cAMP in CHO-K1 cell lines stably expressing either human,dog, cynomolgus monkey, rat, or mouse RXFP1 receptors. In addition, toassess selectivity within the RXFP receptor family, bothRLX-A-AQ1-20KPEG and WT-RLX were also tested for their ability tomodulate cAMP in CHO-K1 cell lines stably expressing the humanreceptors, RXFP2. RXFP3, and RXFP4.

The effect of W T-RLX and RLX-A-AQ1-20KPEG on cellular cAMPconcentration in THP-1 cells, which endogenously express RXFP1, wasdetermined. Based on a 10-point CRC, RLX-A-AQ1-20KPEG was determined tobe 26-fold less potent than WT-RLX with ECs values of 7.6 nM and 0.30nM, respectively (Table 18). The maximal increase in cAMP was notstatistically different between WT-RLX and RLX-A-AQ1-20KPEG. In fact,for all receptors tested, that is, THP-1 and RXFP/CHO-K1 stable lines,both WT-RLX and RLX-A-AQ1-20KPEG were highly similar in their ability tomaximally stimulate cAMP generation. The percent differences in maximalstimulation between these two ligands ranged from 83 to 99% (Table 19).

Both WT-RLX and RLX-A-AQ1-20KPEG were also tested in hRXFP1/CHO-K1 cellsfor activity. As shown in Table 18, based on average values from 10 to12 independent experiments, WT-RLX was determined to be ˜32-fold morepotent than RLX-A-AQ1-20KPEG against the hRXFP1 receptor, with averageECs values of 0.10 and 3.2 nM, respectively.

In order to ascertain selectivity, the activities of WT-RLX,RLX-A-AQ1-20KPEG were determined for the three additional members of thehuman RXFP receptor family (RXFP2, 3, and 4). Activity was determined byeffect on cAMP concentration in RXFP/CHO-K1 stable lines. Whereappropriate activity of the endogenous ligands was also determined.

The agonist activities of RLX-A-AQ1-20KPEG, WT-RLX, and INSL3 againsthuman RXFP2 are shown in Table 18. All three ligands showed similaractivity with regard to maximal stimulation of cAMP. INSL3 generated anEC₅₀ value of 0.46 nM which is highly similar to the 0.58 nM ECs valuereported in the literature. The EC₅₀ value for WT-RLX (5.5 nM) was foundto be ˜10-fold less potent than INSL3 and was reasonably similar to the19 nM EC₅₀ value previously reported in the literature. When compared toWT-RLX, RLX-A-AQ1-20KPEG was determined to be 32-fold less potentagainst human RXFP2 with an EC₅₀ value of 174 nM.

The agonist activities of RLX-A-AQ1-20KPEG, WT-RLX, and H3-RLX againsthuman RXFP3, a G_(αi) coupled receptor, are also shown in Table 18.H3-RLX generated an EC₅₀ value of 0.6 nM for inhibition offorskolin-induced cellular cAMP, which is highly similar to the 0.4 nMEC₅₀ value reported in the literature. In agreement with previousreports, even when tested at very high concentrations (? 3000 nM), bothWT-RLX and RLX-A-AQ1-20KPEG were essentially inactive against the humanRXFP3 receptor.

The agonist activities of RLX-A-AQ1-20KPEG, WT-RLX, and H3-RLX againsthuman RXFP4, a G_(αi) coupled receptor, are also shown in Table 18.Although it had only partial agonist activity (˜40% maximal inhibitionof forskolin-induced cAMP), H3-RLX was relatively potent and generatedan EC₅₀ value of 2.8 nM which is highly similar to the 1.1 nM EC₅₀ valuereported in the literature. In agreement with a previous report in whichporcine WT-RLX was utilized, even when tested at very highconcentrations (≥3000 nM), both human WT-RLX and RLX-A-AQ1-20KPEG wereessentially inactive against the human RXFP4 receptor.

The activities of WT-RLX and RLX-A-AQ1-20KPEG were determined forcynomolgus monkey, dog, rat, and mouse RXFP1. As was done for the humanreceptor, each of these species RXFP1 receptors were stably expressed inCHO-K1 cells to allow for direct comparisons in relative potencies.These results are expected to aid in the understanding of results andfindings for in vivo pharmacodynamic, efficacy, and toxicological animalstudies. Additionally, these results indicate that RLX-A-AQ1-20KPEG maypotentially be efficacious in veterinary medicine. The breadth ofspecies tested, including multiple mammalian orders (primates,carnivora, rodentia) indicate potential broad applicability fortreatment of mammals.

The agonist activities of RLX-A-AQ1-20KPEG and WT-RLX against cynomolgusRXFP1 are shown in Table 18. The EC₅₀ values for WT-RLX andRLX-A-AQ1-20KPEG were 0.06 and 1.97 nM, respectively. When compared toeach other, WT-RLX was 33-fold more potent against the cynomolgus RXFP1receptor than RLX-A-AQ1-20KPEG. Both ligands showed similar activitywith regard to maximal stimulation of cAMP stimulation.

The agonist activities of RLX-A-AQ1-20KPEG and WT-RLX against dog RXFP1are also shown in Table 18. Both ligands showed similar activity withregard to maximal stimulation of cAMP. The EC₅₀ values for WT-RLX andRLX-A-AQ1-20KPEG were 0.56 and 15.0 nM, respectively. When compared toeach other, WT-RLX was 27-fold more potent against the dog RXFP1receptor than RLX-A-AQ1-20KPEG.

The agonist activities of RLX-A-AQ1-20KPEG and WT-RLX against rat RXFP1are also shown in Table 18. Both ligands showed similar activity withregard to maximal stimulation of cAMP stimulation. The EC₅₀ values forWT-RLX and RLX-A-AQ1-20KPEG were 0.03 and 1.1 nM, respectively. Whencompared to each other, WT-RLX was 32-fold more potent against the ratRXFP1 receptor than RLX-A-AQ1-20KPEG.

The agonist activities of RLX-A-AQ1-20KPEG and WT-RLX against mouseRXFP1 are also shown in Table 18. Both ligands showed similar activitywith regard to maximal stimulation of cAMP. The EC₅₀ values for WT-RLXand RLX-A-AQ1-20KPEG were 0.10 and 2.1 nM, respectively. When comparedto each other, WT-RLX was 22-fold more potent against the mouse RXFP1receptor than RLX-A-AQ1-20KPEG.

To further demonstrate ligand selectivity and better understandpotential safety issues, RLX-A-AQ1-20KPEG was tested for binding and/oractivation of GPCRs including adenosine receptors, adrenergic receptors,cannabinoid receptors, dopamine receptors, histamine receptors,muscarinic receptors, opiate receptors, and serotonin receptors.RLX-A-AQ1-20KPEG was inactive against all receptors tested atconcentrations up to or greater than 3500 nM (data not shown).

TABLE 18 Summary of results and relative potencies derived from RXFPcAMP cell-based assay data. cAMP Assay EC₅₀ (nM) Human Human Human HumanCyno Dog Rat Mouse Ligand THP-1 RXFP1 RXFP2 RXFP3 RXFP4 RXFP1 RXFP1RXFP1 RXFP1 WT-RLX 0.30 ± 0.18 0.10 ± 0.02 5.5 ± 1.4 Inactive Inactive0.06 ± 0.02 0.56 ± 0.08 0.03 ± 0.01 0.10 ± 0.02 (>3000) (>3000) RLX-A-7.6 ± .2.1  3.2 ± 0.90 174 ± 66  Inactive Inactive 1.97 ± 0.48 15.0 ±2.9  1.1 ± 0.5 2.1 ± 0.4 AQ1- (>3000) (>3000) 20KPEG INSL3 0.46 ± 0.04H3-RLX 0.60 ± 0.14 2.8 ± 1.5 RLX-A- 1.0 54 Inactive Inactive 1.6 4.70.34 0.66 AQ1- 20KPEG Potency Ratio (hRXFP1- 1.0) Potency 26 32 32 33 2732 22 Ratio (RLX- A-AQ1- 20KPEG/ WT-RLX)

TABLE 19 Maximal cAMP stimulation by RLX-A-AQ1-20KPEG compared to WT-RLX(percent difference). RLX-A- AQ1- 20KPEG/ WT-RLX THP-1 hRXFP1 hRXFP2hRXFP3 hRXFP4 AVG 82.8 94.0 99.0 Inactive Inactive STDEV 5.2 6.4 4.1Inactive Inactive RLX-A- AQ1- 20KPEG/ cyno dog rat mouse WT-RLX RXFP1RXFP1 RXFP1 RXFP1 AVG 90.2 88.9 99.4 97.0 STDEV 4.1 6.9 3.9 3.3

One of the most striking features of the in vitro cAMP data is thatregardless of cell-type (e.g., THP-1 vs. hRXFP1/CHO-K1), receptorspecies ortholog, or member of RXFP family, the relative potencies ofRLX-A-AQ1-20KPEG and WT-RLX remained remarkably constant (Table 18). Forall comparisons, RLX-A-AQ1-20KPEG was determined to be approximately30-fold (ranging from 22 to 33) less potent than WT-RLX. In addition torelative potency, the maximal induction of cAMP was similar for bothligands for all receptors tested, with percent differences ranging from83 to 99%. In total, these data strongly indicate that despite theaddition of a 20-kDa PEG moiety and two amino acid substitutions,RLX-A-AQ1-20KPEG behaves in a manner that is remarkably similar toWT-RLX, excluding the loss in potency. This observation holds trueregardless if one compares receptors for which the ligands were active(e.g., hRXFP1 & hRXFP2) or inactive (e.g., hRXFP3 & hRXFP4), or whenreceptors were expressed endogenously (THP-1) or through recombinantmanipulation (stable hRXFP/CHO-K1 cell lines). In further support ofthis observation, the EC₅₀ values for WT-RLX reported here for hRXFP1and hRXFP2/CHO-K1 cells (0.10 and 5.5 nM, respectively) are in closeagreement with data derived from cAMP-induction assays using HEK-293Tcells stably expressing hRXFP1 and hRXFP2 (EC₅₀ values=0.27 and 19 nM,respectively). The observed continuity amongst all data suggests thatthe 30-fold loss in potency in these in vitro cell-based experimentsshould be taken under strong consideration when either selecting dosesor evaluating PK/PD relationships for in vivo studies.

Methods

The RXFP cDNAs, RXFP family members, as well as species orthologs, wereobtained from commercially available cDNA clones or amplified fromtissue RNAs as follows. Human RXFP1: A cDNA clone was obtained fromOpenBiosystems (MHS4426-99239031. Human RXFP2: A cDNA clone fortranscript variant 1 was obtained from OpenBiosystems(OHS4559-99620528). Cynomolgus monkey RXFP1: The cDNA was cloned out ofMauritian cyno brain RNA by RT-PCR using the following primers: forward:5′-CAACCACCGTGAGCTGTATG-3′ (SEQ ID NO: 37); reverse:5′-CCACAGTATTCTCTGTGAAGAAATG-3′ (SEQ ID NO: 38). Dog RXFP1: The cDNA wascloned out of dog brain RNA by RT-PCR using the following primers:forward: 5′-TCAGTTGCTCAGAAAGAAGGAAATG-3′ (SEQ ID NO: 39); reverse:5′-TGTAGAAATGAGTTTCAGAG-3′ (SEQ ID NO: 40). Rat RXFP1: The cDNA wascloned out of Sprague Dawley rat brain RNA by RT-PCR using the followingprimers: forward: 5′-ATTAATCAGTTGCTCAGAAAGAAGG-3′ (SEQ ID NO: 41);reverse: 5′-CTTGAGAATAAGTGGTTTCCAAAGCC-3 (SEQ ID NO: 42)′. Human RXFP3:A Gateway entry clone in vector pDONR221 was obtained from Invitrogen(IOH28381). Human RXFP4: A Gateway entry clone in vector pDONR201 wasobtained. Mouse RXFP1: A Gateway entry clone in vector pDONR223 wasobtained from OpenBiosystems (OMM4760-101549000). Using standardprocedures (Gateway LR recombination), the RXFP entry clones weretransferred into expression vector pEF-DEST51 (Invitrogen). In contrastto the all other RXFP clones, human RXFP4 was transferred into pcDNA3.1(Invitrogen). For each construct, the RXFP cDNA, as well as its properinsertion into the expression vector, was verified by DNA sequencing.

The coding sequence for each of these was PCR amplified with primerscontaining attB recombination sites and subcloned into the Gateway entryvector pDONR221 using BP clonase enzyme. Receptor gene symbols and lociare as shown in Table 20.

TABLE 20 Receptors introduced into CHO-K1 cells for use in cAMP assays.Gene Species symbol Locus Number Human RXFP1 LOC59350 Human RXFP2LOC122042 Human RXFP3 LOC51289 Human RXFP4 LOC339403 Cynomolgus RXFP1NONE Dog RXFP1 LOC475479 Rat RXFP1 LOC295144 Mouse RXFP1 LOC381489

CHO-K1 cells (ATCC #CRL-9618) were prepared for stable line generationby propagating at 37° C. with 5% CO₂ in F-12 plus GlutaMAX-I medium(GIBCO #31765) supplemented with 10% fetal bovine heat-inactivated serum(Invitrogen #16140), and 1× Antibiotic-Antimycotic (GIBCO #15240-096).CHO-K1 cells were transfected with 12 μg of the appropriate expressionconstruct plasmid using the FuGENE® 6 reagent (Roche #11814443 001) andthe protocol provided by the manufacturer. One day post-transfectioncells were replated into medium containing 10 μg of Blasticidin S HClper mL (GIBCO #A11139-02). After 14 days of Blasticidin S HCl selection,readily distinguishable colonies (approximately 15-20 total) wereisolated using sterile cloning disks (Scienceware #F37847-0001) whichwere subsequently placed into 48-well plates. Stable transfectants weremaintained in the medium containing 10 μg of Blasticidin S HCl per mL.Medium was changed every 2-3 days and cells were grown to confluence.The cells were then trypsinized, counted and plated on PDL-treated96-well transparent black-walled plates at 10,000 cells per well in 150μl medium the day prior to testing for relaxin-dependent activation inthe cAMP assay.

Cell clonal isolates were tested for cAMP induction in response toWT-RLX addition. For the cAMP assay, cells (10,000 cells/well) wereplated into 96-well plates and cultured overnight at 37° C., 5% CO₂. Onthe following day, the medium was removed from the wells and the cellswere stimulated with the addition of ligand [10 nM recombinant humanrelaxin-2 (WT-RLX), R&D Systems #35% RN] in 50 μL buffer [1×HBSS, 5 mMHEPES (7.2), 0.5 mM IBMX, 0.1% BSA]; incubation was for 10 min at 37° C.Unstimulated cells were utilized as reference controls.

Reactions were terminated and cellular cAMP levels were measured usingthe Homogeneous Time Resolved Fluorescence (HTRF) Dynamic 2 cAMP kit(CisBio, #62AM4PEC) according to the manufacturer's protocol. Plateswere analyzed using an EnVision 2104 Multilablel Reader (PerkinElmer).Signal was expressed in terms of the ratio of fluorescence intensity @665 nm/fluorescence intensity @ 620 nm×10,000. The concentration of cAMPin the sample was determined by comparison with standard curvesgenerated from known concentrations of cAMP. Stable expression celllines were selected based on ability of WT-RLX to generate a robustsignal in the cAMP assay.

Testing of RLX-A-AQ1-20KPEG and WT-RLX in cAMP CHO-K1 cell-based assayswas performed essentially as described for the identification of clonalisolates. Ten-point concentration response curves (CRC) were performedin duplicate and utilized to determine relative potencies of ligands.The data were analyzed using GraphPad PRISM 5.0 software. Effectiveconcentration 50 (EC₄)) values, the point at which the response wasone-half maximal, were calculated using a four-parameter logisticequation that allowed for a variable-slope. Each ligand was utilized asits own comparator to determine maximal induction of cAMP accumulation.However, for all species RXFP1 and RXFP2 receptors, the maximal amountof cAMP induction was quite robust (typically 20 to 30-fold abovebaseline) and similar for both WT-RLX and RLX-A-AQ1-20KPEG, as well asthe endogenous ligand for RXFP2 (insulin like 3 protein—INSL3; Cat. No.:4544NS, R&D Systems). THP-1 cells (ATCC Cat. #TIB-202) endogenouslyexpress RXFP1 receptor and upon stimulation with Relaxin exhibit anincrease in intracellular cAMP levels. For the cAMP assay, THP-1 cells(20,000 cells/well) were plated into 96-well plates and culturedovernight in RPMI1640 medium (Cat. No. 22400, Invitrogen) containing 10%FBS at 37° C. 5% CO₂. On the following day, the medium was removed fromthe wells and the cells were stimulated with 2 μM Forskolin (Cat. No.:F6886, SIGMA) and the addition of ligand in 50 μl buffer (1×HBSS, 5 mMHEPES, pH 7.2 to 7.5, 0.5 mM IBMX, 0.1% BSA); incubation was for 20 minat 37° C., 5% CO₂. Non-stimulated cells were utilized as referencecontrols. The human RXFP3 and hRXFP4 receptors are coupled to G_(α1) anda similar HTRF-based protocol as described above was utilized to measureagonist-dependent reduction in forskolin-induced cellular cAMPgeneration. More specifically, cells (10,000 cells/well) were platedinto 96-well plates and cultured overnight at 37° C., 5% CO₂. On thefollowing day, the medium was removed from the wells and the cells werestimulated with 4 μM Forskolin (Cat. No.: F6886, SIGMA) and the additionof ligand (recombinant human relaxin 3, was kindly provided by Dr. JohnWade, Howard Florey Institute, The University of Melbourne, Australia)in 50 μl buffer (1×HBSS, 5 mM HEPES, pH 7.2 to 7.5, 0.5 mM IBMX, 0.1%BSA); incubation was for 25 min at 37° C. Non-stimulated cells wereutilized as reference controls, cAMP measurements and EC₅₀ values weregenerated as described above. H3-relaxin, the endogenous ligand forRXFP3 and RXFP4 receptors, was utilized as a positive control fordetermination of receptor activity.

Example 45

In Vitro Anti-Fibrotic Activity of PEG-Relaxin

This example characterizes the anti-fibrotic activity of a modifiedrelaxin, specifically RLX-A-AQ1-20KPEG in a primary human cardiacfibroblast model.

In the healthy heart, collagen serves an important role in maintainingnormal cardiac architecture and contractile function. In the failedheart, maladaptive changes of increased collagen and cardiac fibrosisoccur which can lead to left ventricular dysfunction. In response topathological stimuli such as AngII and TGF-β, cardiac fibroblastsdifferentiate into myofibroblasts whose phenotype is characterized by,among other changes, increased expression of contractile (e.g., smoothmuscle α-actin) and extracellular matrix proteins (e.g., collagen typeI). Multiple investigators have utilized isolated cardiac fibroblasts(primarily from rat and more recently from human) to investigate themechanisms by which AngII and TGF-β mediate the pro-fibrotic process.Primary rat cardiac fibroblasts stimulated with TGF-β were utilizedpreviously to demonstrate robust antifibrotic effects of WT-RLX. In asimilar manner, we utilized primary human cardiac fibroblasts treatedwith TGF-β to demonstrate for the first time that hRXFP1 is highlyexpressed in these cells and that both WT-RLX and RLX-A-AQ1-20KPEGgenerate robust antifibrotic effects. Relaxin treatment reduced theexpression of α-actin and collagen type I by TGF-β treated cardiacfibroblasts, indicating inhibition of fibrosis.

Initial concentration range (0.1 to 10 ng/mL) experiments using primaryhuman cardiac fibroblasts revealed that TGF-β at 1 ng/mL (24 hrincubation) generated a near-maximal response with regard to inductionof both SM α-actin and collagen type I mRNAs (data not shown). Thus,this TGF-β concentration was utilized for subsequent studies in whichRLX-A-AQ1-20KPEG was compared to WT-RLX.

In untreated primary human cardiac fibroblast cells, a qRT-PCR analysisrevealed raw CT values for the L30 house keeping gene, hRXFP1 and hRXFP2were 14, 20, and 37, respectively. These values indicate that whileexpression of hRXFP1 is relatively high, that of hRXFP2 is extremelylow. Without intent to be limited by theory, these results suggests thatall (or at least a majority) of the effects related to relaxintreatment, are likely mediated by activation of the hRXFP1 receptor.

Treatment with TGF-β at 1 ng/mL (24 hr incubation) resulted in about a 2to 3-fold increase in expression of SM α-actin and collagen type ImRNAs. Additionally, although hRXFP1 mRNA levels remained relativelyhigh, TGF-β treatment (1 ng/mL; 24 hr) resulted in a consistent decrease(˜50-70%) in expression relative to untreated cells (data not shown).

As depicted in FIG. 21A-21D, WT-RLX and RLX-A-AQ1-20KPEG were testedover a broad concentration range between about 0.01× and 100× relativeto their EC₅₀ values as determined in the hRXFP1/CHO-K1 cAMP assays inthe preceding example. TGF-β treatment alone resulted in about a 2 to2.5-fold increase in the expression of collagen type I and about a 2 to3.5-fold increase in the expression of α-actin. As shown in FIGS. 21Aand 21C, both WT-RLX and RLX-A-AQ1-20KPEG generated similarconcentration-dependent decreases in collagen type I expression whencompared to TGF-β only treatment, with maximal inhibition (≥70%)observed for the highest concentrations tested (1 and 30 nM,respectively, corresponding to 100×). A similar pattern oftreatment-dependent amelioration of the TGF-β response was also observedfor SM α-actin expression (FIGS. 21B and 21D).

Notably, the 30-fold potency shift between WT-RLX and RLX-A-AQ1-20KPEGobserved for the RXFP CHO-K1 studies was also maintained in the humancardiac fibroblast assay. Moreover, the similarity of responses withrespect to the magnitude of changes was maintained over a very broadrange of ligand concentrations. Thus, these data, much like the CHO-K1cell-based data, strongly indicate that RLX-A-AQ1-20KPEG behaves in apredictable manner when compared to WT-RLX. Moreover, a possible plateaufor maximal RLX-A-AQ1-20KPEG dependent reduction in the two pro-fibroticgenes was approximately 30 nM. Additionally, there was no evidence of aU-shaped dose response.

These results demonstrate a potent and robust antifibrotic activity forRLX-A-AQ1-20KPEG in primary human cardiac fibroblasts stimulated withTGF-β.

Methods

Primary Human Cardiac Fibroblasts (NHCF-V) were purchased from Lonza,Fairlawn, NJ (Cat. #: CC-2904). NHCF-V cells were isolated from theventricle of normal, adult heart tissue. Cells stained positive forsmooth muscle α-actin and expressed ≥90% collagen I and ≤10% VonWillebrand factor VIII. Cells at passages 3 through 6 were used for thereal-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR) studies. Cells were cultured in FGM™-3 BulletKit™ (Cat. #:CC-4526, Lonza) containing 10% FBS complete medium. At confluence, cellswere split and cultured in 6-well plates at a density of 150,000/wellfor 24 hr in complete medium. Cells were subsequently changed toserum-free medium (Cat. #: CC-3131, Fibroblast Basal Medium, Lonza) for24 hr, then treated with WT-RLX (Cat. #: 3596-RN, R&D Systems) orRLX-A-AQ1-20KPEG in the presence of 1 ng/mL of TGF-β (Cat. #: 240-B, R&DSystems) for another 24 hours. Relaxin treatment concentrations were asindicated in Table 21 below.

TABLE 21 Concentrations of WT-RLX and RLX-A-AQ1-20KPEG used in primarycardiac fibroblast assays. “1X” was set to approximately the EC₅₀ valuefor each form of relaxin, as determined using hRXFP1/CHO-K1 cells in thepreceding example. Treatment Concentration WT-RLX RLX-A-AQ1-20KPEG0.01X   0.001 nM 0.03 nM 0.1X  0.01 nM 0.3 nM  1X 0.1 nM 3 nM 10X 1 nM30 nM 30X 3 nM 90 nM 100X  10 nM 300 nM

Alter treatment, cells in 6-well plates were harvested or total RN Aextraction and qRT-PCR analysis. Total RNA was extracted using theRNeasy Mini Kit (Cat. #: 74106, Qiagen) according to the manufacturer'sprotocols including the on-column removal of genomic cDNA. Real-timeqRT-PCR was performed in a two-step manner; cDNA synthesis and real-timedetection were carried out in a PTC-100 Thermal Cycler (MJ Research) andan ABI Prism 7700 Sequence Detection System (Applied Biosystems),respectively. TaqMan Universal PCR Master Mix (Applied Biosystems) wasused in subsequent PCR reactions according to the manufacturer'sprotocols. All qRT-PCR reactions were performed in duplicate.Sequence-specific primers were designed using Primer Express Version 2software (Applied Biosystems) based on published sequences(http://www.ncbi.nlm.nih.gov). Expression levels were normalized tohousekeeping genes GAPDH or L30. The qRT-PCR ΔCT levels for GAPDH or L30did not differ significantly between treatment conditions: thus, theywere expressed at constant levels between samples. Data were analyzedfor significance by two-way ANOVA followed by a Bonferroni's multiplecomparison test using GraphPad Prism Software, version 5.0.

Example 46

Acute Rat Pharmacodynamic Model of Plasma Osmolality Lowering

Sprague-Dawley rats were dosed with RLX-A-AQ1-20KPEG (IV, 0.2 mg/kg) andthe PK profile determined. Three rats were bled at each time point andserum samples were analyzed by the ELISA method below. Serumconcentrations of sodium and osmolality were also measured and relatedto serum levels of drug. For comparison purposes, a second study wasconducted in which vehicle only was dosed to the rat. Three rats wereincluded for both drug and vehicle treated groups. Although the datawere somewhat variable, which was most likely due to the fact that thebaseline was determined by a single data point (time point zero), whenRLX-A-AQ1-20KPEG was dosed to rats by IV bolus (0.2 mg/kg), there was aclear trend for a decrease in serum sodium that was not observed for thevehicle treated animals. In spite of the relatively high variability, asshown in FIG. 22A, there was a 3.9% decrease in serum sodiumconcentration for the 24 hr time point that reached statisticalsignificance (P=0.05). This 3-4% reduction in serum sodium is similar tothe results described in Example 43 above (see FIGS. 20C-20F). Althoughstatistical significance was not reached, a similar trend was alsoobserved for drug-dependent decreases in serum osmolality (FIG. 22B),with maximal lowering observed between 7 and 24 hrs post-dose. Meanserum levels of RLX-A-AQ1-20KPEG at the 4, 7, and 24 hr time points were239, 101, and 8 nM, respectively. Additionally, these levels representapproximately 4, 2, and 0.1-fold the plasma level of relaxin reportedfor rat during the early stages of pregnancy (˜12 ng/mL or 2 nM), whenthe 30-fold shift in potency of RLX-A-AQ1-20KPEG is factored into thecomparison.

In this example both drug and serum electrolyte levels were measured,allowing for a direct analysis of the PK/PD relationship. Experimentalnoise was likely increased by the use of only a single time point toestablish baseline serum electrolyte levels and relatively small samplesize. Nonetheless the data support the conclusions from the precedingexamples and suggest that high levels of RLX-A-AQ1-20KPEG observed atthe early time points (e.g., <1 hr post-dose) do not diminish themaximal pharmacodynamic response observed at later time points (e.g., 4to 24 hr post-dose).

Notably, for the 24-hr time point, when sodium levels were maximallylowered (−3.9%), the mean serum level of RLX-A-AQ1-20KPEG was 8 nM (48ng/mL). This concentration is far below the 239 nM (1433 ng/mL) serumconcentration of RLX-A-AQ1-20KPEG measured at the 4 hr time point whendecreases in serum sodium first reached statistical significance (−3.2%;P<0.05). This suggests that changes in serum sodium occur over a wideconcentration range of drug, and/or that relaxin's effect on serumsodium levels is long-lasting. In support of the latter, it waspreviously reported in similar studies in which WT-RLX was dosed to ratsvia osmotic pump, that upon cessation of infusion, plasma osmolalityremained significantly decreased at 12 hr but was restored by 24 hr(Danielson and Conrad, J Appl Physiol, 2003. 95(4): p. 1509-14). Infurther support of this possibility, in another study conducted byapplicants (data not shown), rats were repeatedly dosed withRLX-A-AQ1-20KPEG over 10 days (1.0, 5.0, 15 mg/kg, qd, sc), and bloodsampling on day 11 (24-hr post last dose) revealed that serum sodiumlevels were significantly (P<0.005) reduced (˜3%) for all doses whencompared to vehicle-treated rats.

In summary, these results further indicate that RLX-A-AQ1-20KPEGgenerated a pharmacodynamic response in a manner highly consistent withthe data reported for WT-RLX. When the ˜30-fold reduction in receptorpotency was accounted for, the drug levels at which these responsesoccurred was also consistent with data reported for WT-RLX.

Methods

ELISA Assay to Measure RLX-A-AQ1-20KPEG in Serum

Concentrations of RLX-A-AQ1-20KPEG in animal serum were measured by anelectrochemiluminescence (ECL) method on a Meso Scale Discovery (MSD)platform. The assay comprised of four incubation steps: (1) overnightcapture antibody coating, (2) blocking for 2 hours, (3) overnight sampleand biotinylated detection antibody incubation, and (4)Sulfo-TAG-labeled streptavidin incubation for 1 hour. A wash step usingPBS containing 0.05% Tween-20 was performed between each incubationstep. On the first day, MSD high-bind plates (MSD, Gaithersburg, MD)were coated with mouse anti-PEG IgM mAb (Cat. #AGP4-PABM-A, lot #SMA-27,Academia Sinica, Taipei, Taiwan) overnight at 4° C. On the second day,plates coated with the capture antibody were blocked with I-Block buffer(0.2% I-Block/PBS/0.1% Tween-20) for 2 hours at 22° C. The test sampleswere thawed at room temperature, mixed well and analyzed at 5% minimumrequired dilution in 0.2% I-Block/PBS/0.1% Tween-20/5% normal CD-1 mouseserum buffer with additional dilutions in neat serum if needed. Qualitycontrols (QCs) and calibrators were prepared using the same lot ofRLX-A-AQ1-20KPEG as the one used in studies. Prepared samples. QCs andcalibrators, together with a biotinylated rabbit anti-human relaxinpolyclonal Ab, were incubated overnight at 4° C. to allow the formationof binding sandwich on the plates. On the third day, the capturedRLX-A-AQ1-20KPEG was detected using Sulfo-TAG-labeled streptavidin (Cat.#R32AD-1, lot #W0013923S, MSD, Gaithersburg, MD) for 1 hour at 22° C.Following addition of MSD read buffer (MSD, Gaithersburg, MD), theluminescence intensity was measured with an MSD Sector Imager 2400 (MSD.Gaithersburg, MD). Standard curves and QCs were evaluated usingacceptance criteria for accuracy and precision of <20%.

TABLE 22 Serum sodium and osmolality in RLX-A-AQ1-20KPEG treated rats.Percent change in serum sodium and osmolality vs. baseline are shown,along with mean serum level of RLX-A-AQ1- 20KPEG at each time point (asdetermined in the preceding example) as the serum concentration andnormalized to the rat pregnancy level (12 ng/mL) and scaled by therelative potency (30-fold), e.g., at 7 hrs, 101 nM × 5.997 ng/mL/nM =605.7 ng/mL; 605.7/30 = 20.2 ng/mL (30X potency shift); 20.2 ng/mL/12ng/mL = 1.7 (rounded to 2). Mean serum Fold difference Serum sodiumSerum Time RLX- relative to rat mean osmolality (hr) A-AQ1- pregnancy %change mean % change post- 20KPEG level and from baseline from baselinedose (nM) potency (±SEM) (±SEM) 0.03 674 11  0.9 ± 0.9  1.9 ± 0.7 0.25584 10 −0.2 ± 1.5 3.1 0.5 540 9 −1.1 ± 1.3 −1.4 ± 1.7 1 456 8 −0.3 ± 2.0−0.3 ± 1.4 2 392 7 −2.5 ± 1.3 −0.5 ± 1.1 4 239 4 −3.2 ± 1.0 −0.5 ± 1.2 7101 2 −3.6 ± 1.6 −2.4 ± 1.4 24 8 0.1 −3.9 ± 1.2 −2.3 ± 1.7 36 3 0.1 −1.3± 1.4 −0.9 ± 1.5 48 1 0.01 −0.7 ± 1.4 −1.4 ± 1.4 72 0.2 0.003 −0.2 ± 1.2−0.1 ± 1.6 76 0.1 0.002 −0.2 ± 1.2  0.5 ± 2.0

Example 47

Mouse Isoproterenol Heart Failure Model of Cardiac Fibrosis

This model describes development of a rodent model of heart failure (HF)and assessment of RLX-A-AQ1-20KPEG efficacy in this model system.

Adrenergic stimulation enhances cardiac function, however excessivecatecholamine stimulation can be cardiotoxic and contribute to acute HF.In mice, selective overexpression of the ⊕2-adrenergic receptor (β2-AR)in the cardiac myocyte, as well as chronic administration ofisoproterenol (ISO), are both well-established methods for inducing HF.ISO is a nonselective β-AR agonist (stimulates both p1- & β2-AR, but notα1-AR) that simultaneously increases heart rate, cardiac contractility,cardiac relaxation and vasodilation. Cardiac function and tissueperfusion in response to ISO are both increased by the inotropic effect,and impaired by the systemic hypotension and tachycardia. The net impacton the heart with sustained ISO administration is calcium overload, freeradical generation, impaired carbohydrate metabolism, loss of ATPreserves and impaired mitochondrial calcium handling. Depending on thedose and duration of ISO exposure, a progression from adaptivehypertrophy to fibrotic cardiomyopathy is observed in rodents.

Antifibrotic effects have been observed upon administration of WT-RLX tocardiac-specific β2-AR-transgenic mice (Samuel et al., Endocrinology,2004. 145(9): p. 4125-33). However, although there have been numerousstudies employing ISO-HF mice, most of these studies involved the use ofgenetically modified mice. In total, we identified 36 mouse ISO studiesdescribed in the literature where ventricular hypertrophy and/orfibrosis was documented, yet only 4 studies utilized pharmacologicagents. One study of particular interest demonstrated an antifibroticeffect for olmesartan, a member of the ARB drug class that has provenbenefit in HF patients. In this olmesartan study, they measured changesin cardiac levels of collagen mRNA as well as collagen protein contentusing qualitative histology (Zhang et al., J Mol Cell Cardiol, 2007.42(4): p. 804-11).

In this example, HF was induced by administration of ISO, and cardiaccollagen content, quantified by either biochemical (hydroxyproline) orhistological (picrosirius red staining) methods, was utilized as an endpoint to assess the antifibrotic effects of WT-RLX and RLX-A-AQ1-20KPEGin ISO-infused mice.

Mice were continuously infused with ISO using an osmotic minipump (30mg/kg/day, sc) for 12 days. Treatment groups consisted of Vehicle(co-administered vehicles for WT-RLX and RLX-A-AQ1-20KPEG), WT-RLX (0.5mg/kg/day, osmotic minipump) and RLX-A-AQ1-20KPEG (1.2 mg/kg/day, sc,qd) with test article dosing initiated concurrent with ISO dosing(methods are further described below).

The WT-RLX dose was expected to achieve a physiologically-relevantplasma exposure of between 10 to 30 ng/mL. The RLX-A-AQ1-20KPEG dose wasselected to produce a 30-fold greater plasma exposure over WT-RLX toaccount for reduced potency of the PEGylated peptide (300 ng/mL based onmiddle of peak/trough). Serum concentrations determined at the end ofthe study were 40±4 ng/mL for WT-RLX (N=8) and 2322±212 ng/mL (N=6) forRLX-A-AQ1-20KPEG, representing a 58-fold difference (˜2-fold higher thanthe expected 30-fold difference). Two vehicle-treated mice died prior totermination of the study. There were no differences among treatmentgroups in heart weight, body weight, or the ratio of heart weight tobody weight. Clinical chemistry values also did not differ amongtreatment groups, including serum sodium levels. The absence of asignificant reduction in serum sodium levels in the ISO-infused mousetreated with WT-RLX or RLX-A-AQ1-20KPEG is thought to reflect dominanceof the adrenergic pathway over osmotic regulation of serum electrolytesvia a centrally-mediated mechanism.

For all treatment groups, a quantitative blinded histological analysiswas utilized to assess percent cardiac collagen content (collagenarea/total tissue area). The level of percent cardiac collagen contentin the vehicle-treated ISO-infused mice was 4.88±0.77% (N=6, mean±SEM).This represents an approximate 5-fold increase compared to the1.09±0.18% (N=10, mean±SEM) cardiac collagen content observed forsham-treated mice in a separate pilot HF study in which quantitativehistological analysis was also utilized (data not shown). When comparedto Vehicle, treatment with WT-RLX resulted in a significant 39%reduction (P<0.05, N=8) in collagen content. Treatment withRLX-A-AQ1-20KPEG resulted in a 24% reduction in collagen contentcompared to vehicle (P=0.18, N=8). Though the 0.05 significance levelwas not achieved, the high magnitude of the observed beneficial effecton collagen content and near-significant p-value indicate a trend towardsignificance, i.e., that one would expect statistically significantresults to be obtained with a larger study. Results are showngraphically in FIG. 23 .

Given the severity of fibrosis that was induced with ISO treatment, thedata from this initial study are quite promising. Both RLX-A-AQ1-20KPEGand WT-RLX treatments greatly decreased the level of fibrosis asindicated by collagen content. Although only WT-RLX reached statisticalsignificance, RLX-A-AQ1-20KPEG exhibited a clear trend for reduction incardiac collagen content. Whereas the β2-AR-transgenic mouse stimulatesthe adrenergic pathway exclusively in the cardiac myocyte, systemiccirculation of ISO would be expected to stimulate both β1- andβ2-AR-receptors present throughout the entire body. Activation ofβ1-AR-receptors in the heart will stimulate both positive inotropy andchronotropic, whereas activation of β2-AR-receptors in the vasculaturewill cause SM relaxation and vasodilation (note that ISO does notactivate α1-AR which would result in a dominant vasoconstrictiveresponse in the SMC). It is possible that some of the benefit of WT-RLXin decreasing cardiac fibrosis (−58%) in the β2-AR-transgenic mouse mayhave come from decreasing afterload through vasodilation and reductionin SVR. Thus, it is possible that in the mouse ISO-infusion model,decreases in SVR due to increased circulating levels of ISO bluntedWT-RLX-induced vasodilation and hence further reduction in afterload andcardiac fibrosis.

Methods

Dosing regimens of ISO, WT-RLX and RLX-A-AQ1-20KPEG in the current studywere described in Table 23. Briefly, 11-week old, C57BL/6 male mice wereanesthetized with 2-3% isoflurane and infused with ISO (Sigma, St Louis,Mo; dissolved normal saline containing 0.02% ascorbic acid) at the doseof 15 mg/kg/day for 12 days via a osmotic mini-pump (DURECT, Cupertino,CA) implanted subcutaneously. Upon ISO-containing mini-pumpimplantation, mice were randomized into 3 groups (n=8 per group) andreceived vehicle (10 mM sodium citrate, 150 mM sodium chloride, pH 6.5)or WT-RLX (0.5 mg/kg/day) via a second osmotic mini-pump simultaneouslyfor 12 days. In addition, a second vehicle (10 mM sodium citrate, 150 mMarginine, pH 5.4, se) and RLX-A-AQ1-20KPEG (1.2 mg/kg/day, sc) wasadministered once-daily for 12 days.

TABLE 23 Dosing regimens of WT-RLX and RLX-A-AQ1-20KPEG study inISO-infusion model. Mini-pump Mini-pump Vehicle RLX-A-AQ1- Group #1 (sc)#2 (sc) #2 (sc) 20KPEG (sc) 1 ISO Vehicle #1* Vehicle #2** N/A (15mg/kg/day) 2 ISO WT-RLX Vehicle #2** N/A (15 mg/kg/day) (0.5 mg/kg/day)3 ISO Vehicle #1* N/A 1.2 mg/kg/day (15 mg/kg/day) *10 mM sodiumcitrate, 150 mM sodium chloride (pH 6.5); **10 mM sodium citrate, 150 mMarginine (pH 5.4); N/A: not applied

Animal body weight was measured at baseline (day 0) and terminal (day12) time point. At the end of 12 days, mice were euthanized by terminalbleeding under anesthesia. Heart was rapidly removed, blot-dried onfilter paper, weighed and fixed with 10% neutral buffered formalin.Serum and urine samples were collected at 4 hr post final dose forexposure and clinical chemistry analysis.

For WT-RLX analysis, rat anti-human relaxin monoclonal antibody (R&DSystems, Minneapolis, MN) was immobilized on a Meso Scale Discovery(MSD) high-bind plate as a capture molecule. Samples, calibrators, andQCs brought up to a final matrix of 5% C57BL/6 mouse serum wereincubated on the plates. Samples were analyzed at 5% minimum requireddilution in 0.2% I-Block/PBS/0.1% Tween 20/5% normal CD-1 mouse serumbuffer with additional 1-fold to 27-fold dilutions in neat C57BL/6 mouseserum. The unbound material was washed away and the captured WT-RLX wasdetected using biotinylated rabbit anti-human relaxin polyclonalantibody followed by Sulfo-TAG-labeled streptavidin (MSD, Gaithersburg,MD). Following addition of MSD read buffer, the concentration of WT-RLXin mouse serum samples was calculated from luminescence intensity asmeasured by an MSD reader using a 5-parameter logistic (5-PL)calibration curve generated from WT-RLX calibrators.

Serum levels of sodium, osmolality, blood urea nitrogen (BUN),creatinine, glucose, alanine amino transferase (ALT), urine levels ofsodium, microprotein (MTP) and creatinine were determined using ADVIA1800 clinical chemistry analyzer (Siemens Healthcare Diagnostics,Tarrytown, NY).

The following morning after collection of hearts into 10% neutralbuffered formalin, the hearts were positioned in a heart matrix and cuttransversely at mid-ventricle into two equal halves. The two halves wereplaced in labeled tissue cassettes. Tissue cassettes were loaded intothe Leica ASP300 automatic tissue processor and the “Mouse Tissue”program was run in series: 10% neutral buffered formalin (2 hr); 70%,80%, 95%, 95% ethanol (20 min each); 100%, 100%, 100% ethanol (15 mineach); xylene (2×20 min); paraffin 1, 2, 3 (30 min each).

All steps were conducted at room temperature, except paraffin (60° C.),and under vacuum. After processing, the hearts were embedded in paraffinblocks using the Shandon embedding center. Both halves of the heart wereoriented within the same paraffin block. Sections were cut at athickness of 6 μm on the Shandon Finesse rotary microtome. Sections werefloated on a 42° C. water bath, and then picked up on “plus” microscopeslides. The slides were left to dry on a slide warmer at 50° C.overnight.

The sections were stained with picrosirius red to demonstrate collagen.The staining protocol was run on the Sakura DRS automatic stainer in theseries: xylene (2×1 min); 10%, 70%, 40% ethanol (1 min each); distilledwater (1 min); picrosirius red (1 hr); acetic acid water (2× rinse);distilled water (1 min); 40%, 70%, 100% ethanol (1 min each), xylene(2×1 min). Picrosirius red contained 0.5 mg Sirius Red (Sigma, DirectRed 80) in 500 ml saturated aqueous picric acid. Acetic acid watercontained 5 ml acetic acid in 11 ml distilled water. Stained sectionswere coverslipped on the Sakura SCA automatic coverslipper and then leftto dry for a minimum of 30 minutes.

Mid-ventricle heart sections were measured for collagen content usingthe BioQuant image analysis system. Color thresholds were set todifferentiate collagen (red) from the rest of the heart tissue(yellow/orange). For each sample a total of 20 fields of view at amagnification of 20× were randomly selected around the left ventricle.Each field was measured for total tissue area followed by collagen area.A percentage of fibrosis was then calculated. All analysis was conductedon tissue sections that had been assigned random numbers. Note that inpreliminary studies, coronal sections of 3 hearts obtained fromISO-treated mice revealed that the mid-ventricle section was optimal forfibrosis assessment in this model.

Statistical analysis was performed using Student's t test or One-wayANOVA and post-hoc Dunnett's test. P<0.05 was considered statisticallysignificant. Prism 5.0 software (GraphPad Software, La Jolla, CA) wasused for all calculations.

While the invention has been described by way of examples and preferredembodiments, it is understood that the words which have been used hereinare words of description, rather than words of limitation. It isunderstood that the examples and embodiments described herein are forillustrative purposes only and that various modifications or changes inlight thereof will be suggested to those of ordinary skill in the artand are to be included within the spirit and purview of this applicationand scope of the appended claims. Changes may be made, within thepurview of the appended claims, without departing from the scope andspirit of the invention in its broader aspects. Although the inventionhas been described herein with reference to particular means, materials,and embodiments, it is understood that the invention is not limited tothe particulars disclosed. The invention extends to all equivalentstructures, means, and uses which are within the scope of the appendedclaims.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

What is claimed is:
 1. A composition for intravenous or subcutaneousadministration to a human subject in need thereof, comprising: (1) abiologically active modified relaxin polypeptide, wherein: (a) saidmodified relaxin polypeptide comprises a relaxin A chain polypeptide anda relaxin B chain polypeptide, wherein said relaxin A chain polypeptidehas a sequence at least 95% identical to SEQ ID NO: 4, and said relaxinB chain polypeptide has a sequence at least 95% identical to SEQ ID NO:6, and said non-naturally encoded amino acid is substituted in said Achain polypeptide at residue 1; and (b) said non-naturally encoded aminoacid is linked to a linker or polymer, wherein said non-naturallyencoded amino acid comprises a first functional group and the linker orpolymer comprises a second functional group, wherein the firstfunctional group and second functional group are not identical and eachcomprise a carbonyl group, an aminooxy group, a hydrazide group, ahydrazine group, a semicarbazide group, an azide group, or an alkynegroup, and the resultant covalent linkage created by the reaction of thefirst and second functional groups comprises a triazole or an oximelinkage; and (2) a pharmaceutically acceptable carrier and/or excipient.2. The composition of claim 1, wherein the non-naturally encoded aminoacid is selected from a para-substituted, ortho-substituted, ormeta-substituted phenylalanine comprising a carbonyl group, an aminooxygroup, a hydrazide group, a hydrazine group, a semicarbazide group, anazide group, or an alkyne group, or wherein said non-naturally encodedamino acid comprises para-acetyl-L-phenylalanine.
 3. The composition ofclaim 1, wherein the modified relaxin polypeptide exhibits an increasedin vivo half-life by at least 2-fold relative to the human relaxinpolypeptide of SEQ ID NO: 4 and SEQ ID NO:
 5. 4. The composition ofclaim 1, wherein said non-naturally encoded amino acid is linked to alinker and said linker is linked to a polymer, a water-soluble polymer,a polymer comprising poly(ethylene glycol), a label, a dye, a fattyacid, a carbohydrate, a saccharide, a cyclodextrin, a fluorophore, orbiotin.
 5. The composition of claim 1, wherein said relaxin A chainpolypeptide has the sequence of SEQ ID NO: 4 containing saidsubstitution of said non-naturally occurring amino acid at residue 1 andsaid relaxin B chain polypeptide has the sequence of SEQ ID NO:
 6. 6.The composition of claim 1, which comprises an amount of saidbiologically active modified relaxin polypeptide effective for treatmentof a fibrotic disorder.
 7. The composition of claim 6, wherein saidfibrotic disorder is selected from cardiac fibrosis, pulmonary fibrosis,renal fibrosis, hepatic fibrosis, coronary fibrosis, bone marrowfibrosis, dermatological fibrosis, and a fibrotic eye disorder.
 8. Acomposition for intravenous or subcutaneous administration for treatingheart failure in a human subject in need thereof, comprising: (1) abiologically active modified relaxin polypeptide, wherein: (a) therelaxin polypeptide comprises a relaxin A chain polypeptide and arelaxin B chain polypeptide, wherein said relaxin A chain polypeptidehas a sequence at least 95% identical to SEQ ID NO: 4, and said relaxinB chain polypeptide has a sequence at least 95% identical to SEQ ID NO:6, and said non-naturally encoded amino acid is substituted in said Achain polypeptide at residue 1; and (b) said non-naturally encoded aminoacid is linked to a linker or polymer by a triazole or an oxime linkage;and (2) a pharmaceutically acceptable carrier and/or excipient.
 9. Thecomposition of claim 8, wherein said non-naturally encoded amino acid islinked to said linker or polymer by an oxime linkage resulting from areaction of a first functional group and a second functional group,wherein said first functional group comprises a carbonyl group and saidsecond functional group comprises an aminooxy group.
 10. The compositionof claim 8, wherein said non-naturally encoded amino acid comprises saidfirst functional group and said second functional group is linked tosaid linker or polymer, and the oxime linkage resulting from thereaction of said first functional group and said second functional grouplinks said non-naturally encoded amino acid to said linker or polymer.11. The composition of claim 8, wherein the non-naturally encoded aminoacid is selected from a para-substituted, ortho-substituted, ormeta-substituted phenylalanine comprising a carbonyl group, an aminooxygroup, a hydrazide group, a hydrazine group, a semicarbazide group, anazide group, or an alkyne group, or wherein said non-naturally encodedamino acid comprises para-acetyl-L-phenylalanine.
 12. The composition ofclaim 8, wherein the modified relaxin polypeptide exhibits an increasedin vivo half-life by at least 2-fold relative to the human relaxinpolypeptide of SEQ ID NO: 4 and SEQ ID NO:
 5. 13. The composition ofclaim 8, wherein said non-naturally encoded amino acid is linked to alinker and said linker is linked to a polymer, a water-soluble polymer,a polymer comprising poly(ethylene glycol), a label, a dye, a fattyacid, a carbohydrate, a saccharide, a cyclodextrin, a fluorophore, orbiotin.
 14. A composition for intravenous or subcutaneous administrationfor treating heart failure in a human subject in need thereof,comprising: (1) a biologically active modified relaxin polypeptide,wherein: (a) the relaxin polypeptide comprises a relaxin A chainpolypeptide and a relaxin B chain polypeptide, wherein said relaxin Achain polypeptide comprises the amino acid sequence of SEQ ID NO: 4substituted with said non-naturally encoded amino acid at residue 1, andsaid relaxin B chain polypeptide comprises the amino acid sequence ofSEQ ID NO: 6; and (b) said non-naturally encoded amino acid is linked toa linker or polymer by a triazole or an oxime linkage; and (2) apharmaceutically acceptable carrier and/or excipient.
 15. Thecomposition of claim 14, wherein said non-naturally encoded amino acidis linked to said linker or polymer by an oxime linkage resulting from areaction of a first functional group and a second functional group,wherein said first functional group comprises a carbonyl group and saidsecond functional group comprises an aminooxy group.
 16. The compositionof claim 14, wherein said non-naturally encoded amino acid comprisessaid first functional group and said second functional group is linkedto said linker or polymer, and the oxime linkage resulting from thereaction of said first functional group and said second functional grouplinks said non-naturally encoded amino acid to said linker or polymer.17. The composition of claim 14, wherein the non-naturally encoded aminoacid is selected from a para-substituted, ortho-substituted, ormeta-substituted phenylalanine comprising a carbonyl group, an aminooxygroup, a hydrazide group, a hydrazine group, a semicarbazide group, anazide group, or an alkyne group, or wherein said non-naturally encodedamino acid comprises para-acetyl-L-phenylalanine.
 18. The composition ofclaim 14, wherein the modified relaxin polypeptide exhibits an increasedin vivo half-life by at least 2-fold relative to the human relaxinpolypeptide of SEQ ID NO: 4 and SEQ ID NO:
 5. 19. The composition ofclaim 14, wherein said non-naturally encoded amino acid is linked to alinker and said linker is linked to a polymer, a water-soluble polymer,a polymer comprising poly(ethylene glycol), a label, a dye, a fattyacid, a carbohydrate, a saccharide, a cyclodextrin, a fluorophore, orbiotin.